This part is from the bacteriophage M13 genome, bp 3086-3132 in M13K07. It directs transcription of M13 gene VII (BBa_M13507, BBa_M13007). Its identity as a promoter was described in Gene (1980) 11:129-148 based on -10,-35 homology and fragment of genome able to bind RNAP.
New England Biolabs M13K07. Sequenced in 2002 by L. Panganaban.
This RBS is part of the bacteriophage M13 genome, initiating translation of the gene VII message (BBa_M13007). The part aligns with base pairs 1092-1107 in M13K07 genome. It was identified as the RBS for gene VII message by Wezenbeek et al in Gene (1980) 11: 129-148
position 3&5 mutated promoter lux pR-3/5 (luxR & HSL regulated)
The position 3&5 mutated promoter lux pR-3/5 is a product of site-directed mutagenesis of promoter lux pR (BBa_R0062). The promoter lux pR is based on the Vibrio fischeri quorum sensing gene promoters. The position 5 mutated promoter lux pR-3/5 gives a lower expression of downstream genes in an autoinduction system. In another word, the lux pR-3/5 has a lower efficiency than the lux pR(BBa_R0062). Similar to promoter lux pR, the expression is up-regulated by the action of LuxR protein combined with the signal molecule HSL.
We have generated two point mutants by site-directed mutagenesis from the sequence of lux pR(BBa_R0062). In these mutants, the G residue of position 5 was changed to a C and the C residue of position 3 was changed to a T respectively.
Promoter Family Member with Hybrid Operator (D001O17)
Promoter Family Member with Hybrid Operator (D001O61)
This part is from the bacteriophage M13 genome, bp 786-835 in M13K07. It directs transcription of M13 gene V (BBa_M13505, BBa_M13005). Its identity as a promoter was described in Gene (1980) 11:129-148 based on -10,-35 homology and fragment of genome able to bind RNAP.
Prlux-rbs-rfp-Ter. It produces rfp when there is enough luxR and AHL.
T1-motni, terminator for Aspergillus niger
T1-motni is terminator that works in the fungus Aspergillus niger
Compatible with the Plug'n'Play assembly standard
PCR using primers with biobrick prefix and surfix, and then cloned into pSB1AC3.
This is the 1500 bp upstream of the yeast PGK1 coding region. This is a putative constitutive promoter.
Source: Saccharomyces cerevisiae genomic DNA.
This part was obtained from yeast genomic DNA.
Anderson Promoter with lacI binding site
Promoters of the Anderson Promoter Collection with lacI binding site derived from [http://parts.igem.org/Part:BBa_R0010 R_0010] to make them [http://openwetware.org/wiki/IPTG IPTG] iducible.
The "lacI binding site" of Part [http://parts.igem.org/Part:BBa_R0010 R_0010] differs from lacO from Part [http://parts.igem.org/Part:BBa_J33207 BBa_J33207] although they should be same. We choosed part BBa_R0010 because we assumed older parts would be reliable.
Assambly of [http://parts.igem.org/Part:BBa_J23100 J23100] and lacI-binding-site of [http://parts.igem.org/Part:BBa_R0010 R_0010]
optimized (TA) repeat constitutive promoter with 15 bp between -10 and -35 elements
Scissors - Primary plasmid (part 1) [LasR generator]
Part of the iGEM2009 Team:Southampton rock-paper-scissors 'scissors' primary plasmid.
Constitutive promoter selected to allow for continuous expression of C0179.
See relevant basic biobrick parts (J23119, B0034, C0179, B0015)
Promoter Family Member (U073O11+D062O22)
We choosed lacI-binding-site of part BBa_R0010 because we assumed older parts would be more reliable. The part is shipped in backbone [http://parts.igem.org/Part:BBa_J61002 J61002] because the functionality is directly visible by adding IPTG. RFP ([http://parts.igem.org/Part:BBa_E1010 E1010]) should get expressed then.
Assambly of [http://parts.igem.org/Part:BBa_J23102 J23102] and lacI-binding-site of [http://parts.igem.org/Part:BBa_R0010 R_0010] The "lacI binding site" of Part [http://parts.igem.org/Part:BBa_R0010 R_0010] differs from lacO from Part [http://parts.igem.org/Part:BBa_J33207 BBa_J33207].
pBAD is an E.coli promoter that is induced by L-arabinose. K206000 is a mutagenized pBAD promoter that is responsive to lower concentrations of arabinose than wild type ( I13453 ) and, additionally, exhibits a higher maximum of protein expression as measured by coupling to a fluorescent reporter.
There were no special design considerations.
The sequence was obtained by applying all mutations that upregulated AraC binding and subsequent promoter activity listed in reference [1].
Rick Carrick, Ambhi Ganesan, Alex McMillan
This promoter was isolated by taking 500bp upstream of the MFA1 gene of S. cerevisiae. This promoter will be activated when A factor is expressed in an A haploid yeast cell.
This promotor is on the crick strand of the entered sequence.
Promoter Family Member (U049O11+D002O22)
Arabinose induced promoter. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
BBa_K091146 promoter (PAI+LasR inducible & AI+LuxR repressible) controls the expresion of LasI enzyme, no terminator
The biobricks BBa_K091146 and BBa_S03154 where used to build this biobrick
modified Lutz-Bujard LacO promoter,with alternative sigma factor σ24
Lac O and sigma 24 binding conserved sequences
Bidirectional, with the reverse estimated to be less effective than the forward. Has a polyA tail of 6 residues.
This part was designed for the Plug'n'Play with DNA assembly standard (RFC 80), therefore this part contains tails at the 5' and 3' ends as shown on the figure below.
Promoter sequence for constituitive expression
Rock-paper-scissors - Scissors secondary plasmid [las pR, LacI generator]
iGEM2009 Team:Southampton rock-paper-scissors 'scissors' secondary plasmid.
Regulated expression of LacI (C0012) by Las pR (R0079).
See relevant basic biobrick parts (R0070, B0034, C0012, B0015).
There is a G->T point mutation of pSpac at the -1 site, changing the promoter to a stronger one - hence the name pSpac-hy, hy meaning hyper. This promoter drives transcription at rates of about ten times that of the wild type (pSpac) promoter.
Comes from the pPL82 vector, which is intended as a B. subtilis integrational vector. It is placed in front of the MCS in the plasmid, as it would drive the expression of the gene cloned into the MCS both in B. subtilis and E. coli.
HasR is the membrane-bound receptor in the Has system, a heme acquisition system originally from the Serratia marcescens. The HasR protein is involved in binding with a heme-loaded HasA hemophore to trigger downstream HasS and HasI. It has been shown that the N terminal of HasR is especially crucial for signal transduction. With the heme-loaded hasA bound to hasR, it induces a conformational change in HasR that causes the activation of HasS, an anti-sigma factor. HasS then releases HasI (sigma factor) that was initially bound to it and HasI ultimately is able to trigger expression of genes under control of pHas (part number).
The HasS gene is originally found in the Serratia marcescens. We obtained the gene sequence from the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/nuccore/X81195.2) and synthesised the gene.
This part has the arabinose regulated promoter (AraC/pBAD) upstream of a ribosome binding site. This is ideal for inserting a protein to be expressed downstream of this part as it's expression can be regulated by the presence of the concentration of arabinose in the growing environment/media of the cells. ==What this part actually does== This composite part includes the AraC/pBAD promoter and a strong ribosome binding site designed from the Anderson family of RBS. The AraC/pBAD promoter can be used with prokaryotic chassis to regulate expresion of proteins. When the AraC/pBAD promoter is placed upstream of your canditate translational unit (protein to be expressed) it allows control of the trancription of the said protein. This is done via controlling the concentration of arabinose sugar in the environment/solution of bacterial growth. Controlling the transcription will not ensure translation. The ribosome binding site is ideally placed a small distance away from the promoter site to ensure that the ribosome will bind and start translating the protein coding sequence found downstream.
The part was designed with RFC10 assembly from supplied biobricks of the iGEM2009 distribution.
The AraC promoter can be found naturally at 1.52min of the E.coli genome ([http://ecogene.org/geneinfo.php?eg_id=EG10054 EcoGene number EG10054]. The RBS was taken from the designed sequences of RBS by the Anderson family. For more information visit the original part page [http://partsregistry.org/Part:BBa_J61100 here].
Flexible linker used to fuse two proteins.
Designed considering the flexibility and separation needed between our fused proteins.
Promoter from the copper-sensetive CusR/CusS two component signal system. (E.Coli)
Sequence from the NCBI database, with added BioBricks prefix and suffix.
ribosome binding site for creA gene in the E Coli cre operon
modified Lutz-Bujard LacO promoter,with alternative sigma factor σ32
Lac O and sigma 32 binding sites' promoter sequences
Invertible sequence of dna includes Ptrc promoter
Seuqence is inverted by the FimE protein. Inside recognition sequence is a Ptrc promoter sequence. Switch is irreversible.
This promoter is repressed by lacI and luxR with HSL. It contains a lux box and a lacI binding site.
R0010 modified with the insertion of a lux box in between -35 and -10.
T7 promoter with an A->C mutation at the -16 position of the consensus sequence
This part is composed of an arabinose-inducible promoter for superfolded GFP attached by a linker to CpxR (on the N terminus of CpxR).
We had to test both attachments, N and C terminus of the CpxR.
The arabinose promoter and the sfGFP were obtained from the registry, while the CpxR was obtained from the E coli genome.
This biobrick has a promoter inducible by PAI+LasR (BBa_R0079)and produces LuxI enzyme (BBa_F1610). This enzyme produces 3OC6HSL (AI).
The DNA of BBa_R0079 and BBa_F1610 biobricks where used.
This contains CmR, a terminator and then AmpR and a terminator.
Plasmid for Plug'n'Play. Contains an Ampicillin resistance marker.
modified Lutz-Bujard LacO promoter,with alternative sigma factor σ38
Lac O and sigma 38 binding sites conserved sequences
The -35 box of R0065 has been modified to promote constitutive (LuxR+3OC6HSL-independent) gene expression. An extended -10 sequence has also been added to increase absolute transcription. The promoter still contains the operators OR1 and OR2 such that it retains its sensitivity to CI.
B0015+pBAD+vioAB+B0015+pBAD+TPP Up-regulated Hammerhead Ribozyme 1.20+vioD+B0015+pBAD+vioE
B0015+pBAD+vioAB+B0015+pBAD+TPP ribozyme 1.20+vioD+B0015+pBAD+vioE
The Ribosomal Binding Site of vioD is contained in TPP ribozyme 1.20.
The hammerhead ribozyme comes from E. coli, and the TPP aptamer comes from B. subtilis.
LacI-repressible promoter A81 developed by Sidney Cox (http://www.nature.com/msb/journal/v3/n1/full/msb4100187.html). Showed a regulatory range (ON/OFF) of 1.5x10^4.
This part has two fused proteins downstream of an arabinose-inducible promoter: CpxR-IFP[1] and CpxR-IFP[2]. This construct was made in all orientations: - CpxR-IFP[1] + CpxR-IFP[2] (BBa_K1286008) - IFP[1]-CpxR + CpxR-IFP[2] (BBa_K1486010) - CpxR-IFP[1] + IFP[2]-CpxR (BBa_K1486011)
All orientations were made, so all dimerizations could be tested (see main page for biobrick numbers). Flexible linkers were used for each of the fused proteins, and an RBS was put before each one of them.
this hybrid promoter is designed to be consecutively off.It combines with plasR(R0079) and lambda cI OR1(see C0051).Adding P-AI-1 sould activate the promoter, while repressed by lambda cI.
"CI binding of the OR region. It binds OR1 with highest affinity, OR2 with next highest, and OR3 with lowest"(http://www.nonanone.com/qw/files/Biology%204450/test2_lecture8,9.txt)
Promoter, Zif23 regulated, test: between
Promoter with standard -10 and -35 sites. A symmetric Zif23 dimer target site is between the -10 and -35 sites.
consensus sequences based on [Jensen98].
Promoter and ribosome binding site of the Lactococcal nisin inducible operon.
The clp gene from Xanthomonas campestris
c-di-GMP Repressed DNA Binding Protein (Clp) Quorum sensing and bacterial cell signalling are mechanisms known to be utilised by bacteria to regulate their virulence. The RpfC-RpfG sensory system has been found to activate the production of virulence factors like extracellular enzymes and polysaccharides in Xanthomonas campestris. It has been also suggested that this system is involved in linking environmental stimuli, including DSF, with pathogenicity in Xanthomonas campestris. DSF is a cis-unsaturated fatty acid from the DSF family. The RpfC-RpfG two-component system is the most well understood one and is present in all Xanthomonads. http://www.ncbi.nlm.nih.gov/pubmed/?term=r+ryan+dsf+family http://www.ncbi.nlm.nih.gov/pubmed/?term=A+two-component+system+involving+an+HD-GYP+domain+protein+links+cell%E2%80%93cell+signalling+to+pathogenicity+gene+expression+in+Xanthomonas+campestris
The sequence had 1 PstI site that was mutated out by site-directed mutagenesis.
The source of this part is Xanthomonas campestris pv. campestris 8004 genomic DNA which was provided by the Robert Ryan lab in the Molecular Microbiology department of the University of Dundee, UK.
The part contains CpxR with an upstream arabinose-inducible promoter.
The arabinose had to be upstream of the CpxR. Also, the sequence had to be tested because of a mutation, and it works perfectly.
The CpxR comes from the genome of E coli, and the arabinose promoter comes from a biobrick.
Native rbs from crtI (phytoene dehydrogenase) of Pantoea ananatis (formerly Erwinia uredovora) DSMZ 30080 (ATCC 19321). Accession: D90087.
Virtual part to be added to the coding sequence.
Pantoea ananatis (formerly Erwinia uredovora) DSMZ 30080 (ATCC 19321). Accession: D90087.
Design considerations This part was designed for the Plug'n'Play with DNA assembly standard (RFC 80), therefore this part contains linkers at the 5' and 3' ends as shown on the figure below.
Promoter Family Member with Hybrid Operator (D001O27)
Promoter (lambda cI and luxR regulated -- hybrid)
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr
cI repressor negatively regulates this promoter and LuxR activates its transcription.The effect of cI is dominant over LuxR. This part is based on the LuxR and cI repressor regulated hybrid promoter designed and tested by Ron Weiss. It requires the binding of two cI repressor dimers for maximal repression and contains two cI repressor binding sites namely, OR1 and OR2. This promoter is leaky in the sense that 'some' transcription is seen in the absence of both cI and LuxR. LuxI cI activity of promoter + + zero + - maximum - + zero - - leaky (no quantitative information)
This part was designed based on the LuxR and cI repressor regulated hybrid promoter tested by Ron Weiss and the LuxR-LuxICDABE sequence annotated by Tom Knight AF170104 .
Promoter of fliL gene from E. coli flagella
''fliL'' is a class 2 gene that is part of the construction of the flagella ( ''Ordering Genes in a Flagella Pathway by Analysis of Expression Kinetics from Living Bacteria'' - S. Kalir, J. McClure, K. Pabbaraju, C. Southward, M. Ronen, S. Leibler, M. G. Surette, U. Alon, ). Its promoter is consitutively repressed. It is activated by flhD4C2 and by fliA with different forces ( ''Using a Quantitative Blueprint to Reprogram the Dynamics of the Flagella Gene Network'' - Shiraz Kalir and Uri Alon ). A deeper characterization is in project
The primers used to amplify this sequence are : Forward : TCGAATTCGCGGCCGCTTCTAGAGCAAGGGCGTGTAACAGGCAAC Reverse : TCCTGCAGCGGCCGCTACTAGTAGTCATGTGTTGCGGTCTTCCTGTG
This part comes from the genome of ''E.coli'' K12 strain MG1655.
Complex Quorum sensing circuit that receives the signal of PAI+LasR and AI+LuxR to control the production of LuxI and LasI enzymes.
The biobricks BBa_K266006 and BBa_K266000 where used to build this biobrick
Artifical terminator, estimated %T~=80 6bp stem, 4nt loop Bidirectional, estimated reverse %T=40
Bidirectional, with the reverse estimated to be less effective than the forward. Has a polyA tail of 5 residues.
This part has a fused protein downstream of an arabinose-inducible promoter: IFP[1]-CpxR. Biobrick BBa_K1486015 has IFP[2]-CpxR with an arabinose promoter. This construct was made in both orientations: BBa_K1486012 is CpxR-IFP[1] (with an arabinose promoter).
Both orientations were made, as well as for IFP[2] so all dimerizations could be tested (see main page for biobrick numbers). Flexible linkers were used to fuse both proteins, and an RBS was put between the promoter and the cds.
Resistance cassette for selection of mammalian cells. The cassette contains a SV40 promoter, the neomycin resistance gene, and the SV40 poly A as a terminator.
This is a derivate of BBa_K105027. Due to a point mutation in the TATA box region the efficiency of this minimal promoter is only 70 % of the natural yeast CYC1 promoter.
This BioBrick contains the TATA-box with one point mutation and most of the 5'UTR (-139 to -36, relative to the translational start) of the natural CYC1 promoter. A Kozak sequence is not included and has thus to be added! Even if there isn't any need for this BioBrick to be fusible to other protein domains, we stayed with our design.
Amplification and Mutation from the plasmid pAH3
sense TWO INPUTS, activation by AHL and repression by LacI.
Compared with existing BioBrick hybrid promoter R0065, this promoter is different in that its sequence is much shorter with lux box and -35 region overlapping, and that it is repressed by LacI, not cI. Also, the output is clearly detected as shown in Fig. 3.
T7 promoter (strong promoter from T7 bacteriophage)
T7 promoter is very specific promoter which is transcribed only by specific T7 RNA polymerase. Usually this promoter is used in expression systems where T7 promoter is cotransfected with T7 RNA polymerase. That ensures strong transcription of desired genes.
Rock-paper-scissors - Paper secondary plasmid [lux pR, LacI generator]
iGEM2009 Team:Southampton rock-paper-scissors 'Rock' secondary plasmid.
Regulated expression of LacI (C0012) by Lux pR (R0062).
See relevant basic biobrick parts (R0062, B0034, C0012, B0015).
Raissa Estrela Curado, Tais Sineiro Herig and Gleidson Silva Teixeira
nir promoter from Synechocystis sp. PCC6803
The ''nir'' promoter is nitrate inducible.
The ''nirA'' promoter originates from ''Synechocystis'' sp. PCC6803.
LacI-repressible promoter B4 developed by Sidney Cox (http://www.nature.com/msb/journal/v3/n1/full/msb4100187.html). Showed a regulatory range (ON/OFF) of 2.5x10^4.
SpoVG ribosome binding site (RBS) for B. subtilis
Description: SpoVG is an endogenous ribosome binding site from B.subtilis. The sequence of the spoVG ribosome binding site is AAAGGUGGUGA which is complementary to the sequence UUUCCUCCACU from the 3' region of the 16s rRNA from B.subtilis. Previous research showed that the predicted binding energy of the 16s rRNA to the RBS is -19kcal 1
In order to ensure that the RBS is functional the actual ribosome binding site was maintained and the distance between the RBS and the start codon maintained. In order to conform to the biobrick standard the sequence flanking the RBS had to be changed but the distance between the promoter and RBS, and start codon and RBS was maintained.
The sequence was taken from a previous research paper [1] and was constructed by Geneart.
Promoter veg a constitutive promoter for B. subtilis
Pveg is a constitutive promoter that constitutively expresses the P43 protein in ''B.subtilis''. Pveg contains binding sites for the ''B.sutbilis'' major sigma factor#1. Pveg in ''B.subtilis'' utilises two binding sites to cause high expression of genes#2, however our Pveg is lacking the upstream site to give a medium level of gene expression. It has been noted that the sporulation master regulatoion factor spoOA interacts with Pveg though it is not known how#3. The context with which we used the promoter Pveg is as a '''Polymerase Per Second''' (PoPS) generator.
The biobrick part was designed to include a single binding site for the ''B.subtilis major sigma factor. In addition the biobrick standard was applied to the promoter Pveg sequence.
The Pveg promoter was suggested to us by Dr. Jan-Willem Veening of Newcastle University. This sequence supplied was compared to that of the DBTBS database#3 then a section containing the binding site synthesised by Geneart.
LsrA promoter (indirectly activated by AI-2)
The regulatory network for the uptake of Escherichia coli autoinducer 2 (AI-2) is comprised of a transporter complex, LsrABCD; its repressor, LsrR; and a cognate signal kinase, LsrK. This network is an integral part of the AI-2 quorum-sensing (QS) system. Once uptaken into the cell, AI-2 will be phosphorylated under the effect of LsrK. Without the presence of AI-2, LsrA promoter is inhibited by the repressor, LsrR. Phosphorylated AI-2 will prevent LsrR from inhibiting this promoter, hence activating it so that the downstream sequence can be expressed. To prevent the cell from self-inducing this promoter, we must suppress its AI-2 producing capability. It can be done by knocking out LuxS, one of the vital gene required for producing AI-2. By doing that, LsrA promoter can only be activated under the presence of AI-2 from other sources than the host cell which is LuxS mutant (say, by contact with normal cells). More information on AI-2 quorum-sensing (QS) system please refer to: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1952038
Promoter (lacI regulated, lambda pL hybrid)
Neelaksh Varshney, Grace Kenney, Daniel Shen, Samantha Sutton
Inverting regulatory region controlled by LacI (<bb_part>BBa_C0010</bb_part>, <bb_part>BBa_C0011</bb_part>, etc.) The PLlac 0-1 promoter is a hybrid regulatory region consisting of the promoter P(L) of phage lambda with the cI binding sites replaced with lacO1. The hybrid design allows for strong promotion that can nevertheless be tightly repressed by LacI, the Lac inhibitor (i.e. repressor) (<bb_part>BBa_C0010</bb_part>) ([LUTZ97]). The activity of the promoter can be regulated over a >600-fold range by IPTG in E.Coli DH5-alpha-Z1 (same paper reference).
hybrid promoter design to create strong promoter that is, at the same time, highly repressible. note that the upstream operator installed in this hybrid is slightly different than the one in the original source (Lutz and Bujard, 1997). the most upstream operator region is slightly truncated in the represillator version, so that both operators in the hybrid are the same sequence. see references for details. also, the sequence has been truncated after the transcriptional start site. LacI binds to this regulator. This part is incompatible with species containing active LacI coding regions. Lactose and IPTG disable the operation of LacI and increase transcription. This part is incompatible with environments containing lactose or lactose analogs.
represillator of Elowitz and Leibler (2000)
The promoter from the yeast gene CYC1. Activity characterized here: http://dx.doi.org/10.1016/0378-1119(95)00037-7.
From Mumberg, Muller, and Funk p413 pCYC. See ref. above
To edit. Pense ?? rajouter les linkers, je ne sais pas s'ils sont de igem ou s'il faut ajouter la s??quence ?? la registry. Check pour tous tes biobricks et dis-moi, please.
Yeast Optimized sfGFP-C + ADH1 terminator + CaUra3 Cassette
Strong constitutive promoter for Bacillus subtilis
'host-takover module' from B.subtilis Bacteriophage SPO1
PgpdA, promoter for Aspergillus nidulans
Strong constitutive Aspergillus nidulans promoter
From the plasmid p71 from the plasmid collection of the Center for Microbial Biotechnology at the Technical University of Denmark
TetR - TetR-4C heterodimer promoter (negative)
This promoter is repressed by a heterodimer of wild-type TetR and mutant TetR-4C. It differs from wild-type TetR promoter in that the +4 and -4 regions of its O2 binding site have been changed, and the -35 region of the reverse promoter (pA) has been destroyed.
The O2 and O1 mutations are all single point mutations, occuring at the first base which appears listed here.
This part is from the bacteriophage M13 genome. It directs transcription of M13 gene II (BBa_M13502, BBa_M13002). Promoter identified in Gene (1980) 11:129-148 based on -10,-35 homology and fragment of genome able to bind RNAP.
Fabiana de Melo Duarte, Leonardo Minete, Maria Carolina Grassi and Lucas P. Parizzi
Py is the tra operon promoter of F-plasmid. It is repressed by the host nucleoid-associated protein, H-NS and activeted by TraJ protein. Conflicting evidence suggests that TraY can act as both an activator and a repressor. The tra operon encodes F-plasmid proteins (except TraJ and TraM) involved in DNA transfer by conjugation in E.coli. The regions shows intrinsic curvature, which could interfere in regulation. For this reason, the two closer bends upstream promoter region are included.
The mechanism for controlling transcription from Py remains undefined. The Py region contains significant intrinsic curvature, which could interfere in regulation
T7 promoter with an A->T mutation at the -10 position of the consensus sequence
Dual-Repressed Promoter for p22 mnt and TetR
This part is a promoter that is repressed by both p22 mnt and TetR. It can be used to control the response of any gene that follows this promoter. It will be repressed by the presence of either p22 mnt or TetR.
TetR usually binds to two operator sites, one located upstream from -35 and one located between -35 and -10. However, p22 mnt also has two operator sites, one between -35 and -10 and another downstream from -10. Since each had an operator site in between -35 and -10, we had to eliminate the TetR operator site in order to allow the promoter to work. TetR was picked because it has a stronger binding than p22 mnt, and so would be more able to use only one site to bind.
This part was designed by combining sequences from parts C0040 (TetR promoter) and C0072 (p22 mnt promoter).
Gli1 binding sites + MET25 inducible yeast promoter
This gene is on the Watson strand of the entered sequence.
The is a repressable promoter for use in Saccharomyces cerevisiae. It is repressed by the presence of methionine. When unrepresssed, is has a moderate level of activity.
From the Saccharomyces cerevisiae genome, by way of Caroline Ajo-Franklin.
position 3 mutated promoter lux pR-3 (luxR & HSL regulated)
The position 3 mutated promoter lux pR-3 is a product of site-directed mutagenesis of promoter lux pR (BBa_R0062). The promoter lux pR is based on the Vibrio fischeri quorum sensing gene promoters. The position 3 mutated promoter lux pR-3 gives a higher expression of downstream genes in an autoinduction system. In another word, the lux pR-3 has a higher efficiency than the lux pR. Similar to promoter lux pR, the expression is up-regulated by the action of LuxR protein combined with the signal molecule HSL.
In the promoter lux pR's sequence, the C residue of position 3 was changed to a T using PCR method.
The position 3 mutated promoter lux pR-3 is based on promoter lux pR (BBa_R0062).
The Gli1 and Zif268-HIV transcription factor binding sites on the mCYC minimal promoter (in that order.) Transcription regulation comes from Gli1 and Zif268-HIV binding transcription factors.
Constructed in our lab with parts from Caroline Ajo-Franklin and David Drubin.
mutant promoter of lacI gene, which enables high level of lacI protein expression
point mutation made from E. Coli genomic lacI gene promoter
Intermediate designed to accelerate cloning of the HSP promoter ( BBa_K338001 ) by adding an RBS downstream.
Designed to be ligated upstream of the desired heat-shock product. Keep in mind that the promoter is somewhat leaky and that cultures should be grown at 30°C.
The Registry of Standard Biological Parts
The HasA hemophore (19 kDa monomer) is originally from the Serratia marcescens and used in their heme uptake pathway. When bound to heme, the HasA hemophore is able to trigger expression of genes through the Has system. The heme-bound HasA hemophore is able to bind to a membrane receptor HasR, a component of the signalling cascade that regulates expression of the genes in the has operon via HasS and HasI. When HasR is activated, it causes a conformation change in HasS, an anti-sigma factor. This causes the release of the sigma factor, HasI, initially bound to it. HasI then goes on to bind to a specific promoter to trigger expression of genes under the control of that promoter. This is a composite part created from the coding sequence of HasA (BBa K1897000) with the addition of Bba_K525998, which contains a promoter and Ribosome Binding Site (RBS), and Bba_B0015, which contains a double terminator. The addition of these parts make BBa K1897001 a functional unit that can be used for expression, unlike BBa K1897000 which lacks the promoter, RBS and terminator and hence cannot be used on its own for expression.
The presence of a hexa histidine tag would allow for easy protein purification and detection using anti-his antibodies. The promoter BBa_K525998 was chosen as we wished to induce high levels of HasA for purification thus we required a T7 promoter that could be used to induce expression at high levels in E. coli strain BL21(DE3). The terminator BBa_B0015 was chosen as it is commonly used and has a high efficacy.
The HasA coding sequence is originally found in the Serratia marcescens and to construct this part, we used the HasA gene that is from the part BBa K1897000 which was synthesised based on the gene sequence from the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/nuccore/X81195.2). The Promoter, RBS and terminator are biobrick parts that were obtained from the Distribution kits from iGEM.
This is the PLL promoter taken from the P4 phage genome. It is an inducible promoter that is activated by a class of activators, including P2 ogr (I746350), PSP3 pag (I746351) and phiR73 delta (I746352). These different activators should cause different levels of activity of the PLL promoter.
This RBS is part of the bacteriophage M13 genome, initiating translation of the gene II message (BBa_M13002). The part aligns with base pairs 8252-8267 in M13K07 genome. It was identified as the RBS for gene II message by Wezenbeek et al in Gene (1980) 11: 129-148
Compatible with the Plug'n'Play with DNA assembly standard
In native e.coli, this promoter is located upstream of FecA cds. Regulated by FecI protein, which is in turn regulated by FecA.
The lambda t1 is a strong transcriptional terminator with 99% efficiency in vivo.
Promoter Family Member (U049O11+D014O22)
This RBS is part of the bacteriophage M13 genome, initiating translation of the gene III message (BBa_M13003). The part aligns with base pairs 1563-1578 in M13K07 genome. It was identified as the RBS for gene III message by Wezenbeek et al in Gene (1980) 11: 129-148 New England Biolabs M13K07. Sequenced in 2002 by L. Panganaban
argB cassette, selection marker for Aspergillus nidulans
The Aspergillus nidulans gene argB encodes ornithine transcarbamylase and can be used as an auxotrophic marker in the filamentous fungi A. nidulans and A. niger. The successfull use of argB as a selection marker has been documented and described extensively in the literature.
endogenous promoter for Fig1 (Factor Induced Gene 1)
The Rec A(SOS) promoter has been PCR amplified from the chromosomal DNA from MG1655(Ecoli).
A mutant version of the T7lac promoter designed by Dubendorff and Studier. The promoter is based on the strong ??10 T7 promoter but incorporates an A->C mutation at -16. The LacI operator site is a partially symmetric 25bp sequence.
The -16 mutation was included to weaken the consensus promoter as described by Imburgio and co-workers (see BBa_R0183 for more information)
This sequence is based on the T7lac promoter described by Dubendorff and Studier (JMB 1991, 219, 45-59). The only modification is the mutation at -16.
This is a TetR protein generator regulated by the constitutive promoter J23109. The protein has a LVA tag for degradation.
promoter of Paenibacillus sp. WLY78 nif gene cluster
The promoter of Paenibacillus sp. WLY78 nif gene cluster, We named it Pnif. Pnif is a σ70-dependent promoter located upstream of nifB. Pnif is in control of all the gene clusters of nitrogen fixation genome. Confirmation of nif promoter We standardized the nif promoter of Paenibacillus sp. WLY78, and linked it to the upstream of 3 reporter genes (RFP reporter: BBa_K1357010, YFP reporter: BBa_E0430;amilCP reporter: BBa_K1357009) to confirm the reliability of this promoter. The results are shown in Figure 2. A B C http://2015.igem.org/wiki/images/b/b2/Confirmation_of_Pnif_AB.png http://2015.igem.org/wiki/images/thumb/3/34/C-confirmation_of_Pnif.jpeg/622px-C-confirmation_of_Pnif.jpeg Fig.4. Confirmation of nif promoter of Paenibacillus sp. WLY78. (A) Pnif+RFP reporter; (B) Pnif+YFP reporter;(C)Pnif+amilCP reporter. The results confirmed the reliability of Pnif. The initiation of Pnif does not require inducement. Thus it is a effective constitutive promoter.
effeciency of the promoter in E.coli for ordinary parts
Pnif is from Paenibacillus sp. WLY78 genome,located upstream of nifB,the first gene of nitrogen fixation cluster.
T7 promoter for expression of functional RNA
Artificial terminator, estimated %T~=55 * 6bp stem, 4nt loop * Bidirectional, estimated reverse %T~<20
Bidirectional, with the reverse estimated to be less effective than the forward. Has a polyA tail of 4 residues.
I0500 with extra trailing restriction sites: Eco-Xba-I0500-HindIII-NcoI-Spe-Pst
BioScaffold Linker - Removes Stop Codons & scars & replaces with a Gly-Ser linker
This part is part of a family of Bioscaffold - Linkers. This part will help users interested in protein-fusion. The usage of the part will allow in frame assembly of 2(+) proteins by: *Removing the stop codons from the upstream protein. *Removing the scar created by the Assembly Standard 10 (RFC10) construction of 2(+) protein coding sequences. *Maintaining the start codon of the downstream protein. *Adding a flexible (Gly-Ser-Gly) linker between the two protein coding sequences. *Acting as a translational buffer if ligation is incorrect. ===Assembly Instructions=== *Create an Assembly Standard 10 fusion as below ; **Upstream Protein Part - Bioscaffold Linker - Downstream Protein Part** *Use the BioScaffold Specific Enzymes (i)Restrict with BpuEI to remove upstream scar (created between Upstream Protein - Bioscaffold) and stop codons from upstream protein part. (ii)Ligate and you will get the intermediate product; **Upstream Protein (w/out Stop codons) - Tyr - BioScaffold Linker - Downstream Protein** *Restrict with CspCI to remove downstream scar. *Ligate to get the final product; **Upstream Protein - Tyr-Gly-Ser-Gly- Downstream Protein**
This part was designed taking into consideration the requirements by Assembly Standard 10 (RFC10).
This part is completely designed ''de novo''. This sequence does not occur naturally.
Stephen Lee, Roshan Kumar, Joe Levine, Ziyan Chu (Polkadorks, IAP 2004)
Positively regulated, OmpR-controlled promoter. This promoter is taken from the upstream region of ompF. Phosphorylated OmpR binds to the three operator sites and activates transcription.
The promoter includes three OmpR binding sites: F1, F2, and F3. The promoter starts at -107 and goes up to the transcriptional start site at +1. A distant fourth binding site, F4, has been deleted. It is involved in negative regulation of ompF (Head, Tardy and Kenney, 1998), so its deletion is intended to make the promoter solely activating. The efficacy of this promoter versus the ompC upstream region (BBa_R0082) or the truncated ompC upstream region (BBa_R0083) is currently unknown.
Sequence comes from the natural gene 5 RBS. Using gene 5 as a strong RBS is from initial results from experiments characterizing all natural T7 RBSs.
to be used with transcription factor BBa_J34802
This promoter is a modified version of the Mnt promoter that is also responsive to LacI. The promoter should be repressed by Mnt repressor. It should also be repressed by LacI, and in the absence of Mnt repressor, should be induced by IPTG.
Mnt repressor binds as a tetramer to two half-operator sites. Introduction of a LacI binding site between the Mnt promoter -10 sequence and the start site for transcription should allow for repression by LacI. Accordingly, the hybrid promoter was designed by 1) Remove all bases of mnt promoter 3??? to -10: gagtcgtattaattt will be replaced by tgtgtggaattgtga, and 2) Position lacI binding site (composed of an inverted repeat) from the lacI regulated promoter (R0010) immediately 3??? to truncated mnt promoter.
This part comes from the Mnt promoter and the LacI promoter
Monomeric red fluorescent protein (mRFP1) is a derivative of the red fluorescent protein cloned from Discosoma coral (DsRed) (1). The successful use of mRFP1 in Aspergillus nidulans has previously been described (2). This part does not contain a stop codon. {| border="1" !colspan="8"|Spectroscopic data (1) |- |Excitation maximum |Emission maximum |Extinction coefficient per chain |Fluorescence quantum yield |pKa |t0.5 for maturation at 37??C |colspan="2"|Rate of photobleach relative to EGFP |- |584 nm |607 nm |44,000 M-1cm-1 |0.25 |4.5 |<1h |fast 7.2 |slow 0.16 |}
This part was designed for the Plug'n'Play with DNA assembly standard (RFC 80), therefore this part contains tails at the 5' and 3' ends as shown on the figure below. Please notice that mRFP1-module is not the same as BBa_K678030 or BBa_K678032.
From p71-mRFP1 of the Center for Microbial Biotechnology plasmid collection at the Technical University of Denmark
This part is the natural invI RBS from Salmonella Typhimurium LT2.
This part was not designed with consideration for biobrick standard assembly in mind. This RBS has not been explicitly tested for expression level in a fluorescence or B-Gal assay.
constitutive promoter which outputs .3 TIPs point mutants of wild type rrnB promoter P1
RBS.4 (weaker) -- derivative of BBa_0030
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
Weaker RBS based on Ron Weiss thesis. Strengths relative to <bb_part>BBa_B0030</bb_part>, <bb_part>BBa_B0031</bb_part>, <bb_part>BBa_B0032</bb_part>.
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix ("RBS-3" in figure 4-14 of thesis). No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info for this part: Brian Chow
NOT Gate Promoter Family Member (D001O33)
This promoter is designed to bind to C2003.
The placement of the operator was designed to be near optimal for steric interference between C2003 and RNA polymerase.
The expression level of this RBS has not been quantified.
NikR promoter, a protein of the ribbon helix-helix family of trancription factors that repress expre
Nickel responsive transcriptional regulator
Pars promoter from Escherichia coli chromosomal ars operon.
This is the promoter region of the Escherichia coli chromosomal ars promoter. It is repressed by ArsR in the absence of arsenate or arsenite (Cai, J., and DuBow, M.S. 1996. Expression of the Escherichia coli chromosomal ars operon. Canadian Journal of Microbiology 42, 662-671; Diorio, C., Cai, J., Marmor, J., Shinder, R., and DuBow, M.S. 1995. An Escherichia coli chromosomal ars operon homolog is functional in arsenic detoxification and is conserved in Gram negative bacteria. Journal of Bacteriology 177, 2050-2056).
No modifications were made to the sequence. There appears to be an unexpected repeat of ACT immediately before the SpeI site. This does not affect the biobrick suffix but means that the suffix is 5 bases downstream of the annotated transcriptional start site rather than 2 as recommended. The reason for this is unclear.
Escherichia coli K12. Sequence obtained from X80057 (gi:510824). Regulatory sequence annotation from Cai, J., and DuBow, M.S. 1996. Expression of the Escherichia coli chromosomal ars operon. Canadian Journal of Microbiology 42, 662-671; Diorio, C., Cai, J., Marmor, J., Shinder, R., and DuBow, M.S. 1995. An Escherichia coli chromosomal ars operon homolog is functional in arsenic detoxification and is conserved in Gram negative bacteria. Journal of Bacteriology 177, 2050-2056.
eukaryotic -- derived from SV40 early poly A signal sequence
It is cloned in BioBrick vector pSB1AK3.
nhaA promoter, that can be regulated by pH and nhaR protein.
It's E.coli K12 sodium/proton anti-transport promoter. It can be a pH sensor. We test pH senses in different pH value and Sodium concentration.
NOT Gate Promoter Family Member (D001O77)
CpxR is a transciption factor that dimerizes and becomes active when phosphorylated by the transmembrane histidine kinase CpxA. CpxA in turn becomes active when the bacterial cell is submitted to different kinds of shock (pH, temperature, pressure, protein misfolding in the inter-membrane space...).
When making composite parts with CpxR that are supposed to dimerize, we had to take care about the positioning of the other elements regarding the parallel dimerization of CpxR.
The part has been extracted from the E coli genome.
The pLux' promoter is a modified version of BBa_R0062 containing the -35 region TTTAAT in place of TTTACG. Transcriptional activation is expected to occur in the presence of LuxR and AI-1, just as with R0062. The modified -35 region was designed to serve as a control for the evaluation of BBa_K091156, the pLux+ Las- promoter.
This promoter is identical to pLux+ Las- with the exception of the Las box's absence
Lac O and sigma 32 binding sites conserved sequences
This RBS is part of the bacteriophage M13 genome, initiating translation of the gene X message (BBa_M13010). The part aligns with base pairs 480-495 in M13K07 genome. It was identified as the RBS for gene II message by Wezenbeek et al in Gene (1980) 11: 129-148
CinR, CinL and glucose controlled promotor
This promotor is controlled by cinr, cinl and extrcellular glucose concentration. High extracellular glucose concentration will inhibit this promotor through the action of phosphorylated OmpR. Phosphorylated OmpR will bind the OmpR binding site of the promotor and block the movement of RNA polymerase. In contrast, the combined action of CinR and CinL will turn on the promotor. But only when the extracellular glucose level is low so OmpR dephosphorylate and dissociate from its binding site will this promotor become active. In essence, this promotor function as an AND gate, it will turn on if and only if the extracellular glucose level is low and there are some functional CinR and CinL protein inside the cell.
The key to the function of the promotor is the inhibitory ability of OmpR binding site. To ensure maximum affinity with phosphorylated OmpR, we use all three OmpR binding site.s.
strong synthetic E. coli ribosome binding site with SacI site.
This is a strong synthetic E. coli ribosome binding site. It is synthesised as two complementary oligonucleotides rather than being incorporated into a biobrick plasmid. It incorporates a SacI site overlapping the XbaI site, which is preserved when it is added to any other biobrick. This allows easy detection of the RBS after it has been added upstream of a biobrick coding sequence in a plasmid vector.
Note the presence of a SacI site overlapping the XbaI site, which is preserved when this biobrick is added to any other biobrick. At the time of writing, this biobrick is added as a short piece of DNA composed of two complementary oligonucleotides rather than being incorporated into a biobrick cloning vector by itself. It can be added upstream of a biobrick coding sequence, and its presence can easily be detected in miniprep DNA on a gel by using a SacI-SpeI or similar digest.
This part is part of a family of Bioscaffold - Linkers. This part will help users interested in protein-fusion. The usage of the part will allow in frame assembly of 2(+) proteins by: *Removing the stop codons from the upstream protein. *Removing the scar created by the Assembly Standard 10 (RFC10) construction of 2(+) protein coding sequences. *Maintaining the start codon of the downstream protein. *Adding a flexible (Gly-Ser-Gly) linker between the two protein coding sequences. *Acting as a translational buffer if ligation is incorrect. ===Assembly Instructions==== *Create an Assembly Standard 10 fusion giving ; **Upstream Protein Part - Bioscaffold Linker - Downstream Protein Part *Use the BioScaffold Specific Enzymes **Restrict with BpuEI to remove upstream scar (created between Upstream Protein - Bioscaffold) and stop codons from upstream protein part. **Ligate and you will get the intermediate product; Upstream Protein (w/out Stop codons) - Tyr - BioScaffold Linker - Downstream Protein **Restrict with BseRI to remove downstream scar. **Ligate to get the final product; Upstream Protein - Tyr-Gly-Ser-Gly- Downstream Protein
Part was designed taking into account RFC10 assembly standard and as an attempt to be upwards compatible with RFC10.
This part was designed ''de novo''. The sequence does not occur naturally.
Tyr Residue involved in Restriction/Ligation
Promoter driving the rhlI gene in Pseudomonas aeruginosa. Activated by the RhlR+C4HSL. Also likely activated by LasR+3OC12HSL
Genomic sequence of Pseudomonas aeruginosa
Promoter hyper-spank is an inducible promoter that has been designed for high expression in ''B.subtilis''. Gene expression under the promoter hyper-spank can be induced by addition of Isopropyl β-D-1-thiogalactopyranoside (IPTG). The context with which we used the promoter hyper-spank, was to take an input of IPTG and give '''Polymerase Per Second'''(PoPS) as an output. IPTG does not induce the promoter hyper-spank directly, but requires the transcriptional regulator '''LacI''', ( BBa_K413035 ). This means that LacI must be constitutively expressed in ''B.subtilis'' in order to use the promoter hyper-spank.
Biobrick standard was applied to the promoter hyper-spank sequence.
The part was designed using the sequence from the ''B.subtilis'' genome and from previously published papers 123. This sequence was then synthesised by Geneart.
constitutive promoter with (C)12 between -10 and -35 elements
With 12 C repeats between the -10 and -35 elements, the distance between the two elements is optimal for the sigma factor to bind to the promoter. Thus, the promoter should be in the 'on' state.
This part was PCR amplified from two synthesized primers that bound to each other.
The BBa_J23100 constitutive promoter with Lac systems inverter
The biobricks BBa_J23100 and BBa_Q04121 where used to build this biobrick
Lead Promoter (Part 4) messed up EcoRI site
Promoter Family Member (U073O27+D062NUL)
Promoter of flgB gene from E. coli flagella
''flgB'' is a class 2 gene that is part of the construction of the flagella ( ''Ordering Genes in a Flagella Pathway by Analysis of Expression Kinetics from Living Bacteria'' - S. Kalir, J. McClure, K. Pabbaraju, C. Southward, M. Ronen, S. Leibler, M. G. Surette, U. Alon ). Its promoter is consitutively repressed. It is activated by FlhD4C2 and maybe by FliA with different strength ( ''Using a Quantitative Blueprint to Reprogram the Dynamics of the Flagella Gene Network'' - Shiraz Kalir and Uri Alon ). '''Work in progress :''' Deeper quantitative characterization
The primers used to amplify this sequence are : *Forward : GTTTCTTCGAATTCGCGGCCGCTTCTAGAGACAGTATCGCGATGATCGCCACGCTACG *Reverse : GTTTCTTCCTGCAGCGGCCGCTACTAGTAAGCATATCTCCTCCGCAGGTATCAAAATT
This part is a heat shock promoter, it refers to the transcription factor sigma32. when the temperature rise, the transcription factor sigma32 expresses a lot and binds to the RNA ploymerase to recognize the conserved sequence in -35 and -10 region, then the promoter starts to transcript.
The whole sequence of this part is only 90bp, so take a futher consideration if you intend to digest it for use.
We align the HSP promoters from the literature and then find the conserved sequence, then we just modified the promoter of groES of E.coli, and synthesis the part through BIC genescript.
This part is the natural RBS for the sopB gene in the Salmonella Type III Secretion System.
This part was not designed with consideration for biobrick standard assembly in mind. This RBS hasn't been explicitly tested for expression rate in a Fluorescence or B-Gal assay.
This is the consensus RBS for Bacillus subtilis.
The part must be 8 nucleotides away from the first methionine of the coding sequence, and so we added 'TGT' downstream of the actual binding sequence: AAAGGAGG. This, combined with the truncated non-CDS BioBrick prefix scar, gives exactly 8 'spacer bases' between the 3' end of the RBS and the start of the coding sequence.
Dual-Repressed Promoter for p22 cII and TetR
This part was designed by combining sequences from parts R0040 (TetR promoter) and R0053 (p22 cII promoter).
Lactate Permease from Kluyveromyces lactis
Ra??ssa Estrela Curado, Ta??s Sineiro Herig and Gleidson Silva Teixeira
JEN1 from Kluyveromyces lactis is a homologue gene to JEN1 of Saccharomyces cerevisiae. It ecodes a monocarboxylate acids permease shared by lactic and pyruvic acids. Queiros O, Pereira L, Paiva S, Moradas-Ferreira P and Casal M. 2007. Functional analysis of Kluyveromyces lactis carboxylic acids permeases: heterologous expression of KlJEN1 and KlJEN2 genes. Curr Genet 51, 161-169.
This part was amplified by PCR from K. lactis genomic DNA.
ribosome binding site for transcription initiation
Promoter (luxR & HSL regulated -- lux pR)
Promoter activated by LuxR in concert with HSL The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes.This expression is up-regulated by the action of the LuxR activator protein complexed with the autoinducer, 3-oxo-hexanoyl-HSL. Two molecules of LuxR protein form a complex with two molecules of the signalling compound homoserine lactone (HSL). This complex binds to a palindromic site on the promoter, increasing the rate of transcription.
This promoter is based on the Vibrio fischeri quorum sensing gene promoters. Two genes LuxI and LuxR and transcribed in opposite directions as shown below. The original sequence from which the parts <bb_part>BBa_R0062</bb_part> and <bb_part>BBa_R0063</bb_part> were derived is shown in the picture below. <img src="<bb_file>Image1.gif</bb_file>" width="614" height="362">
Promoter gsiB is a sigma factor B dependent promoter found in ''B.subtilis''. In ''B.subtilis'' endogenous sigma factor B is activated under mild stress. These mild stress conditions can be generally split into nutrient stress response and physical stress response. Nutrient stress response is triggered by low levels of ATP and GTP and physical stress response is triggered by exposure to blue light, salt, heat, acid or ethanol1. The promoter gsiB has been used previously as a read out for the activation of sigma factor B 2. The context with which we used the promoter gsiB, was to take blue light as an input and give '''Polymerase Per Second'''(PoPS) as an output. To do this the other potential inputs need to be carefully controlled so that only blue light activated the sigma B and gives a PoPS output. In order to get sufficient sigma B activation by blue light the light receptor YtvA, part...., needs to be over expressed in ''B.subtilis'' 3.
Biobrick standard was applied to the promoter gsiB sequence.
The part was designed using the sequence from the ''B.subtilis'' genome and from previously published papers 23. This sequence was then synthesised by Geneart.
Assambly of [http://parts.igem.org/Part:BBa_J23103 J23103] and lacI-binding-site of [http://parts.igem.org/Part:BBa_R0010 R_0010] The "lacI binding site" of Part [http://parts.igem.org/Part:BBa_R0010 R_0010] differs from lacO from Part [http://parts.igem.org/Part:BBa_J33207 BBa_J33207].
PLRV Coat Protein gene - Isolated from Desir??e potato
This part encodes the major coat protein of the Potato Leafroll Virus (PLRV). It was isolated from a PLRV infected Desir??e potato leaf by iGEM team Wageningen UR 2012. This was done by cDNA synthesis followed by PCR. The PLRV major Coat Protein should be capable of forming a Virus-Like Particle, but this was not (yet) confirmed for this part.
PLRV-infected Desir??e potato leaf (RNA).
This is a promoter designed to be repressed by BBa_C2006: a MBP.Zif268.GCN4 homodimer.
The operator site is positioned to increase the steric hindrance between the repressor and RNAP based on comparing the structures of the repressor bound to DNA and RNAP bound to DNA.
This sequence is derived from BBa_R2000 and the protein structures of Zif268.GCN4(-2) bound to DNA and RNAP bound to DNA.
This is the exact region between the end of the ORF of STE2-W and the beginning of BST1-C
This sequence was taken from the yeast genome
This is the second part of the split Renilla luciferase. To be used with rLuc[1].
Promoter Family Member (U061O11+D026O22)
promoter of Alcaligenes eutrophus PhaP gene
Ray Paul Laureano-Mercado & Mark Kirollos
This part is the coding sequence for the enzyme melamine deaminase, optimized for usage in E. coli. This enzyme eliminates an amine group from melamine to produce ammonia and ammeline and is able to remove another amine group at a rate 10 times slower than the first reaction to bring the compound to ammelide. The enzyme does not react to a measurable extent with this last compound, so it metabolizes melamine by consecutive and predictable deaminations.
The TriA gene???s codons were modified from the original and optimized for E. coli to a Codon Adaptation Index (CAI) of .76. After that, some palindromic and mRNA destabilizing sequences were removed to increase the stability of our DNA during synthesis.
The original gene coding for the protein of interest was isolated from a species of Pseudomonas (http://www.ncbi.nlm.nih.gov/nuccore/11890744).
The sicA promoter is a regulatory element in the Salmonella SPI-1 secretion system. It controls the expression of secreted effector proteins in the natural system and turns on late. Please reference Temme et al, 2008, JMB for exact dynamics of this promoter and associated feedback mechanisms. This promoter only functions in Salmonella strains containing SPI-1.
This part can only be used in salmonella strains.
This hybrid promoter is designed to respond to activation by LuxR when it is bound to AHL and to repression by the phage lambda cI repressor. The repression needs to win over activation.
This part is based on the idea of replacing the region of the Lux promoter downstream of the -35 with the OR2 cI half-operator, added LuxR-10 region. Based on what we have found up to now, promoters which are constructed just by inserting OR1/2 downstream of Lux are all very leaky.
this part comes from LuxR promoter and cI lambda promoter.
AND-type promoter synergistically activated by cI and CRP
Pmom is one of the four late promoters of the Mu bacteriophage. It can be activated by the C protein, which is produced by the middle promoter Pm. Pmom works because of a special configuration of the DNA. It can be used to create a sensitivity tuner alongside the C protein of the same phage. This construction can be used to increase the POPS output of a promoter.
The design of this part had no special considerations
Synthetized from the reported sequence of Pmom promoter from Mu bacteriophage
constitutive promoter with (TA)9 between -10 and -35 elements
With 9 TA repeats between the -10 and -35 elements, the distance between the two elements is not optimal for the sigma factor to bind to the promoter. Thus, the promoter should be in the 'off' state.
GFP targeted to the peroxisomes of Aspergillus nidulans
The promoter region (inclusive of regulator binding sites) of the B. subtilis RocDEF operon
This is the reported promoter region from the Bacillus subtilis RocDEF operon as taken from the DBTBS website (http://dbtbs.hgc.jp/). Base 1 to base 21 contains the binding site for the binding factor, RocR. The sequence, TATGCAAAAGAATTTTGCACT, reportedly contains the consensus sequence for binding of this factor. Base 42 to base 62 contains another binding site for the RocR binding factor, with ATATCAGAATGTTTTTGCACC as the binding consensus for this sequence. Bases 113-135 outline the binding site of the binding factor AhrC (CTTGCATTTATATAAAGGGAAAG). Bases 96 to 135 outline the promoter region for this operon (CTTGATTTGGCACAGAACTTGCATTTATATAAAGGGAAAG).
This sequence only outlines the reported promoter region as ascertained from the online resource described above, and may lack nucleotides not described by the operon analysis used.
Information was obtained from: http://dbtbs.hgc.jp/COG/prom/rocDEF.html.
Based on the AHL regulated BioBrick part R0062 and the previous work on LacI regulated promoter (ref. 1), we designed a new promoter regulated by AHL and LacI. Since this promoter is a kind of a "HYBRID" of the two, we call it HYBRID PROMOTER. Compared with existing BioBrick hybrid promoter R0065, this promoter is different in that its sequence is much shorter with lux box and -35 region overlapping, and that it is repressed by LacI, not cI. Also, the output is clearly detected as shown in Fig. 3.
Ptet-rbs-rfp-Ter. It produces rfp when there is less tetR.
This part is the twin of the non-compatible biobrick BBa_K1486008 rendered compatible by mutating the two illegal restriction sites. However, all experiments were run on the aforementioned biobrick.
Site-directed mutagenesis was used to remove the restriction sites in the sequence.
http://www.addgene.org/browse/article/8177/ which was then mutated by us.
Promoter Family Member (U097NUL+D038O22)
NOT Gate Promoter Family Member (D001O55)
modified Lutz-Bujard LacO promoter,with alternative sigma factor σ28
Lac O and sigma 28 binding sites' conserved sequences
This is a low-level constitutive yeast promoter.
From David Drubin in the Silver Lab at Harvard.
This minimal promoter can be used as a base for designing promoters by adding binding sites upstream.
This promoter sequence is a derivative of part BBa_R0011 which is a -35 and -10 recognition site with two lac operator 1's inserted in the spacer region of the RNAP recognition region and before the -35 RANP recognition region. This part utilizes the UV5 double mutation at the -10 RNAP recognition site to recruit RNAP without the help of a CAP binding region.
The length of the fragment makes it cumbersome to gel purify
UV5 -10 RNAP recognition region double mutation
Zif268-HIV binding sites + ADH1 yeast promoter
The Zif268-HIV transcription factor binding sites on the ADH1 constitutive promoter. Transcription regulation should be constitutive plus regulated by Zif268-HIV-binding transcription factors.
yA (green pigment) from Aspergillus nidulans
The gene yA from Aspergillus nidulans encodes a p-diphenol oxidase (laccase) that converts a yellow pigment precursor to the mature green form. The green pigment is located in the walls of the conidia and protects the spores from damage by ultraviolet light. A yA mutant produces yellow spores.
Promoter Family Member (U061O11+D062O22)
This is the PF promoter taken from the P2 phage genome. It is an inducible promoter that is activated by a class of activators, including P2 ogr (I746350), PSP3 pag (I746351) and phiR73 delta (I746352). These different activators should cause different levels of activity of the PF promoter.
no special considerations seemed necessary
The original source is the P2 phage genome. For our work the DNA came from plasmids, kindly supplied to us by Prof. Calendar, University of California (Berkeley): Bacteriophage PSP3 and fR73 Activator Proteins: Analysis of Promoter Specificities; Julien and Calendar, 1996; JOURNAL OF BACTERIOLOGY, Oct. 1996, p. 5668???5675
This RBS has the modified Shine-Dalgarno sequence, it has a reduced secondary structure relative to using the actual Brink sequence with mixed connective sites flanking it. This RBS is modified from B0071 by reducing the 3' sequence so that the SD to ATG spacing is closer to that which is optimal for E. coli.
Infrared signal reporter (J23119:IFP:J23119:HO1)
Gene sequence is obtained directly from Tsien's lab.
Promoter Family Member (U085O11+D050O22)
Taken from no other than Curious George, this promoter is induced by the presence of banana in the metabolism of any mammal. It is very weak however without the presence of the HngR protein. For transcription to reach significant levels both HngR and banana must be present
Monkeys can be dangerous when aggrevated
his is the first part of the split Renilla luciferase. To be used with fLuc[2].
Native ribosome binding site for crtZ (beta-carotene hydroxylase) from Pantoea ananatis (formerly Erwinia uredovora) DSMZ 30080 (ATCC 19321). Accession: D90087.
A virtual part to be added to the coding sequence blunt (without a scar).
BBa_J23100 constitutive promoter directs the expression of tetracycline repressor (TetR). TetR binds to pTet regulatory region resulting in a ''negative control'' of the production of LuxR. The whole system acts as an ''Inverter'' of Tet system controlling LuxR expression. TetR repression is inhibited by the addition of tetracycline or its analog, [http://openwetware.org/wiki/ATc aTc].
The biobricks BBa_K145201 and BBa_K116640 where used for this biobrick
5' UTR site of alcohol oxidase 1 gene (aox1)
5' UTR site of alcohol oxidase 1 gene (AOX1) from K. pastoris used as a recombination site.
Promoter of flgA gene from E. coli flagella
''fliA'' is a class 2 gene that is part of the construction of the flagella ( ''Ordering Genes in a Flagella Pathway by Analysis of Expression Kinetics from Living Bacteria'' - S. Kalir, J. McClure, K. Pabbaraju, C. Southward, M. Ronen, S. Leibler, M. G. Surette, U. Alon ). Its promoter is consitutively repressed. It is activated by FlhD4C2 and maybe by FliA with different strength ( ''Using a Quantitative Blueprint to Reprogram the Dynamics of the Flagella Gene Network'' - Shiraz Kalir and Uri Alon ). '''Work in progress :''' Deeper quantitative characterization
The primers used to amplify this sequence are : *Forward : GTTTCTTCGAATTCGCGGCCGCTTCTAGAGAGCATATCTCCTCCGCAGGTATCAAAAT *Reverse : GTTTCTTCCTGCAGCGGCCGCTACTAGTAACAGTATCGCGATGATCGCCACGCTACGT
LacO and Sigma 24 binding conserved sequences
Modified lambda Prm promoter (OR-3 obliterated)
Lambda Prm promoter modified to be activated but not repressed by the lambda repressor (cI)
In wild type lambda phage, the OR3 site (-27 to -11) reads "tatcccttgcggtgata" on the sense strand of DNA. To prevent lambda repressor (cI) from binding to this site, the 4th through 10th nt of OR3 were replaced by the 7 nt between OR2 and OR1, "aaatagt" (-57 to -51), which in effect mutates 5 nt of the promoter. These nt were chosen to be about halfway between the -10 and -35 boxes of the promoter. Furthermore, since these nt already act as a spacer in wild-type phage, it is hoped they will not create undesired interactions.
Native ribosome binding site of crtY (lycopene cyclase) from Pantoea ananatis (formerly Erwinia uredovora) DSMZ 30080 (ATCC 19321). Accession: D90087.
This is a virtual part to be added, blunt-ended, to the coding sequence.
Plac-rbs-rfp-Ter It produce rfp when there is enough IPTG or less lacI
This promoter is active at higher temperatures.
This is a high level constitutive promoter from the yeast gene TEF2.
From the yeast genome using promoter definition from: Mumberg, Muller, and Funk: http://dx.doi.org/10.1016/0378-1119(95)00037-7
to be used qith t7 polymerase BBa_J34811
Vikram Vijayan, Allen Hsu, Lawrence Fomundam
Kanamycin resistance cassette for modular BioBricks vector construction.
Promoter Family Member (U097NUL+D062O22)
Promoter Xylose is an inducible promoter that has been designed for high expression in ''B.subtilis''. Gene expression under the promoter xylose can be induced by addition of xylose. The context with which we used the promoter xylose, was to take an input of xylose and give '''Polymerase Per Second'''(PoPS) as an output. Promoter xylose is an inducible promoter that has been designed for high expression in ''B.subtilis''. Gene expression under the promoter xylose can be induced by addition of xylose. The context with which we used the promoter xylose, was to take an input of xylose and give '''Polymerase Per Second'''(PoPS) as an output. Xylose does not induce the promoter xylose directly, but requires the transcriptional regulator '''XylR''', ( BBa_K143036 ) This means that XylR must be constitutively expressed in ''B.subtilis'' in order to use the promoter xylose.
Biobrick standard was applied to the promoter xylose sequence.
The part was designed using the sequence from the ''B.subtilis'' genome and from previously published papers 12. This sequence was then synthesised by Geneart.
PLRV Coat Protein gene under IPTG induced promotor
This part consists of BBa_J04500 (IPTG inducible promotor + RBS) and the coding sequence for the major coat protein of the Potato Leafroll Virus (PLRV). This sequence was isolated from a PLRV-infected Agria potato leaf by iGEM team Wageningen UR 2012. This was done by cDNA synthesis followed by PCR. The PLRV major Coat Protein should be capable of forming a Virus-Like Particle, but this was not (yet) confirmed for this part. This gene was isolated from potato leaf roll virus (PLRV) infected plants. The obtained sequence of the coding gene is 100% identical to several PLRV isolates available in the NCBI database. Expression of a similar viral coat proteins was performed by the iGEM2012 team of Wageningen UR, strongly suggesting that this part can be expressed in E. coli as well. In the figure below, the predicted 3 dimensional structure can be seen. The coat proteins will form a icosahedral shaped Virus Like Particle (VLP) with a radius of about 11,5 nm composed of 180 coat proteins.
The coding sequence for this part was isolated from a total RNA sample from an Agria potato leaf.
improved for transcription factor BBa_J34807 binding
A regulatory part composed of a promotor and an RBS, can be used for both transcription and translation. Just add any protein after it.
J23111 is from constitutive promoter family and B0034 is an RBS based on Elowitz repressilator
The placement of the operator site was designed to be near optimal for steric interference between C2003 and RNA polymerase.
This part has a fused protein downstream of an arabinose-inducible promoter: IFP[2]-CpxR. Biobrick BBa_K1486014 has IFP[1]-CpxR with an arabinose promoter. This construct was made in both orientations: BBa_K1486013 is CpxR-IFP[2] (with an arabinose promoter).
Both orientations were made, as well as for IFP[1] so all dimerizations could be tested (see main page for biobrick numbers). Flexible linkers were used to fuse both proteins, and an RBS was put before each one of them.
Promoter, Standard (lambda cI regulated)
Vinay S Mahajan, Brian Chow, Peter Carr, Voichita Marinescu and Alexander D. Wissner-Gross Drew Endy
[Note: This is the same part as R0051 except that the -10 and -35 sites and spacing have been changed to comply with BBa_S0001]. The cI regulated promoter is based on the pR promtoer from bacteriohage lambda. The promoter has two two DNA binding sites for lambda cI repressor <bb_part>BBa_C0051</bb_part>. cI binding results in repression of transcription. The specific sequence used here is based on the cI repressible promoter used in the Elowitz repressilator (and references therein).
In order to address concerns about the promoter transcribing in the reverse direction, we have removed the -35 and -10 signals responsible for the promoter activity in the reverse direction. (More details needed here! DE, 2/24/03) Modified to comply with BBa_S0001: TTGACA-17N-GATACT Incompatible with host expressing cI repressor.
A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000
This promoter was isolated by taking 500bp upstream of the MF(ALPHA)1 gene of S. cerevisiae. This promoter will be activated when alpha factor is expressed in an alpha haploid yeast cell.
Modified version of B0031 from the Voigt Lab. Not currently available as an independent part (only in a tet inverter).
Ampicillin resistance cassette for modular BioBricks vector construction.
Promoter for expression of genes in mammalian cells
From plasmid pSuperior.puro from ￼￼pSuper RNAi System, OligoEngine
This part promotes the transcription of TyrRS(aminoacyl tRNA synthetase).
E.coli We synthetized it according to the GeneBank.
Right facing promoter (for xylF) controlled by xylR and CRP-cAMP
potential looping feature possibly removed
removed RBS, left promoter, and several xylR binding sites
Promoter ctc is a sigma factor B dependent promoter found in ''B.subtilis''. In ''B.subtilis'' endogenous sigma factor B is activated under mild stress. These mild stress conditions can be generally split into nutrient stress response and physical stress response. Nutrient stress response is triggered by low levels of ATP and GTP and physical stress response is triggered by exposure to blue light, salt, heat, acid or ethanol1. The promoter ctc has been used previously as a read out for the activation of sigma factor B 2. *The context with which we used the promoter ctc, was to take blue light as an input and give '''Polymerase Per Second'''(PoPS) as an output. To do this the other potential inputs need to be carefully controlled so that only blue light activated the sigma B and gives a PoPS output. In order to get sufficient sigma B activation by blue light the light receptor YtvA, part...., needs to be over expressed in ''B.subtilis'' 3. ===Reference=== #1 pmid=16267279 #2 pmid=3100810 #3 pmid=17575448
Biobrick standard was applied to the promoter ctc sequence.
puc BA is the complete LHII transmembrane protein coding region found naturally in Rhodobacter Sphaeroides. LHII complexes form the light-harvesting antennae the funnel energy down to LHI and the reaction center. They are naturally found in the ratio of 3-6.7 LHII per LHI. Data suggests the precence of puc C,D, and E regions also contribute to the formation of a functional protein.
puc A and puc B together create the antennae complex necessary to harvest varying intensities of light. A common problem in bioreactors is the uneven distribution of light between cells nearest the light source and cells farther away from the light source. One way of combating this problem is modify the antenna size of the organism. By truncating the light-harvesting antenna, cells at the exterior of the bioreactor waste less light through Non-Photochemical Quenching, thus allowing photons to penetrate deeper and reach cells in the interior of the bioreactor. This leads to an increase in photosynthetic efficiency and productivity for the entire culture.
PCR applified from plasmid pRKCBC3 provided by Dr. Neil Hunter.
Constitutive weak promoter of lacZ which sequence was changed at position 29 (T->G) in order to make it weaker
Strong transcriptional terminator consisting of a 20 bp stem-loop from Salmonella phage P22.
Overlap extension of synthetic oligonucleotides
FliA is a transcription factor involved in the formation of the flagella in E. coli. The promotor of fliA is induced by FlhDC. FliA seems to have a positive autoregulation on its promotor.
This part comes from the genome of E. coli K12 strain MG1655.
araC-Pbad - Arabinose inducible regulatory promoter/repressor unit
Valentina Herbring, Sebastian Palluk, Andreas Schmidt
This promoter can be induced by arabinose. In presens of arabinose the promotor is active.
Guzman LM, Belin D, Carson MJ, Beckwith J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol. 1995 Jul;177(14):4121-30 Khlebnikov A, Skaug T, Keasling JD, modulation of gene expression from the arabinose-inducible araBAD promoter,J Ind Microbiol Biotechnol. 2002 Jul;29(1):34-7.
This promoter sequence (-80 to +1) is responsive to the transcription enhancer glnG.
Promoter Family Member (U037O11+D014O22)
FadA protein coding sequence for fatty acid degradation
For a E.coli to degrade fatty acid, at least four enzymes ??? FadA, FadB, FadD, FadE and transporter???FadL are needed. Long chain fatty acids are firstly being imported by the transmembrane protein FadL. After FAs get into cells, they will be added a CoA by inner membrane-associated FadD (acyl-CoA synthase). β-oxidation is initiated by FadE(acyl-CoA dehydrogenase), which will convert acyl-CoA into enoyl-CoA. The followed cycles of hydration, oxidation, and thiolytic cleavage are carried out by tetrameric complex consisting of two FadA and two FadB monomers. This part is FadA coding sequence, which has 3-ketoacyl-CoA thiolase activity involving in the β-oxidation of living E.coli. The protein takes a part in the planning system to improve whole efficiency of fatty acid degradation. With appropriate promoters we decided, FadA expression can be under regulation of fatty acid concentration.
Before constructing the final system and testing its function, we constructed three biobrick parts with 3-ketoacyl-CoA thiolase activity (FadA, FadI, S-FadA) as candidates.
bleR cassette, selection marker for Aspergillus nidulans
bleR cassette, antibiotic selection marker for Aspergillus nidulans
PLRV Coat Protein gene - Isolated from Bildstar potato
This part encodes the major coat protein of the Potato Leafroll Virus (PLRV). It was isolated from a PLRV infected Bildstar potato leaf by iGEM team Wageningen UR 2012. This was done by cDNA synthesis followed by PCR. The PLRV major Coat Protein should be capable of forming a Virus-Like Particle, but this was not (yet) confirmed for this part.
Monomeric red fluorescent protein (mRFP1) is a derivative of the red fluorescent protein cloned from Discosoma coral (DsRed) (1). The successful use of mRFP1 in Aspergillus nidulans has previously been described (2).
This part was designed for the Plug'n'Play with DNA assembly standard (RFC 80), therefore this part contains tails at the 5' and 3' ends as shown on the figure below. Please notice that mRFP1-TS is not the same as BBa_K678030 or BBa_K678031.
Dual-Repressed Promoter for LacI and LambdacI
This part is a new construct of a promoter that is dually repressed by either LacI or LambdacI proteins.
LambdacI usually binds to two operator sites, one located upstream from -35 and one located between -35 and -10. By itself, LacI binds downstream from -10. This made the part design very straightforward, since the locations of the operator sites do not overlap at all. In order to complete this part, we simply combined the proper sequences and we had a dually-repressed promoter.
This part was made by combining BioBrick parts R0010(LacI promoter) and R0051 (LambdacI promoter), along with intermediate sequence between these operator sites.
PAI+LasR -> LasI+GFP & AI+LuxR --| LasI+GFP
BBa_K091146 promoter (PAI+LasR inducible & AI+LuxR repressible) controls the policistronic expresion of LasI enzyme and GFP, double terminator
The biobricks BBa_K266005 and BBa_E0840 where used to build this biobrick.
Yeast CLB1 promoter region, G2/M cell cycle specific
Description of CLB1 (from SGD): B-type cyclin involved in cell cycle progression; activates Cdc28p to promote the transition from G2 to M phase; accumulates during G2 and M, then targeted via a destruction box motif for ubiquitin-mediated degradation by the proteasome. More information found here: http://db.yeastgenome.org/cgi-bin/locus.pl?locus=CLB1
Part contains 500 bases upstream from the CLB1 ORF. Is NOT a minimal promoter, and has not been tested for composability.
Yeast consensus sequence from SGD chrVII:703140..703639
This part is from the bacteriophage M13 genome, bp 2716-2764 in M13K07. It directs transcription of M13 gene VI (BBa_M13506, BBa_M13006). Its identity as a promoter was described in Gene (1980) 11:129-148 based on -10,-35 homology and fragment of genome able to bind RNAP.
PmgtCB Magnesium promoter from Salmonella
This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: BBb Standard Description Page
This part has a fused protein downstream of an arabinose-inducible promoter: CpxR-IFP[2]. Biobrick BBa_K1486012 has CpxR-IFP[1] with an arabinose promoter. This construct was made in both orientations: BBa_K1486015 is IFP[2]-CpxR (with an arabinose promoter).
Construct assembled to allow the quantitative analysis of the heat shock promoter, K338001 . Produces GFP in response to low-temperature heat shock.
The type of GFP used in this construct happens to have a very long half life. If growing cultures for fluorescence measurements, grow at 30°C - any higher will activate the promoter, causing GFP to be produced.
The YY1 transcription factor binding sites on the mCYC minimal promoter. All transcription regulation comes from YY1-binding transcription factors.
Made in our lab with parts from Caroline Ajo-Franklin and David Drubin.
Promoter Family Member (U073O11+D026O22)
gentamycin/kanamycin resistance cassette
The aacA-aphD gene from the transposon Tn4001 conveys resistance to both Gentamycin and Kanamycin with a single two-domain resistance protein.
;primers to make Biobrick version of GmR gene cassette (P1012) gtttcttcgaattcgcggccgcttctagagTGGACTTGACTGAGTTTATGGAA, P3aph-F gtttcttcctgcagcggccgctactagtaTCTGGACTTGACTCACTTCCTTT, P3aph-R ;P3aph-R (3' - 5') ;aaaggaagtgagtcaagtccagatactagtagcggccgctgcaggaagaaac
Originally from Tn4001 transposon; sequence M18086. Created from plasmid pISM2062 from Chris Minion, Iowa State University.
Assambly of [http://parts.igem.org/Part:BBa_J23110 J23110] and lacI-binding-site of [http://parts.igem.org/Part:BBa_R0010 R_0010] The "lacI binding site" of Part [http://parts.igem.org/Part:BBa_R0010 R_0010] differs from lacO from Part [http://parts.igem.org/Part:BBa_J33207 BBa_J33207].
a constitutive expression promoter or use in mammalian cells. Ribosome binding site is included.
pTet + RBS + mRFP1 + Transcriptional terminator
This part codifies for a red fluorescent protein (BBa_K081014). It??s regulated by a TetR repressible promoter (R0040).
mRFP: Monomeric red fluorescent protein engineered to enhance photoresistance (BBa_E1010). It was used the part BBa_K081014. ptet: Based in the TetR repressible promoter of operon for resistance against tetracycline (BBa_R0040).
This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. In a Bca1256 plasmid backbone. Our microarray results showed that this gene was upregulated four fold in response to low frequency ultrasound and two fold in response to audible sound. We cloned approximately 400 base pairs upstream from the start codon for the ygeF pseudogene.
Promoter for expression in mammalian cells. The SV40 promoter contains an ori.
Inverting regulatory region controlled by LacI (<bb_part>BBa_C0010</bb_part>, <bb_part>BBa_C0011</bb_part>, etc.) The pLac regulatory region is a 243 base-pair sequence with standard BioBrick prefix and suffix sections on its ends. It contains two protein binding sites: CAP, which is generally present in E.coli and is assocciated with cell health and availability of glucose., and LacI, the Lac inhibitor <bb_part>BBa_C0010</bb_part> which binds in an dimerized cooperative manner to inhibit the transcription of the protein that follows. In the presence of lactose or IPTG, an analog of lactose, LacI is unable to correctly bind and inhibit transcription. This allows <bb_part>BBa_R0010</bb_part> to be used as a inverter or as a detector of lactose or IPTG.
LacI binds to this regulator. This part is incompatible with species containing active LacI coding regions. Lactose and IPTG disable the operation of LacI and this regulator. This part is incompatible with environments containing lactose or lactose analogs.
The Plac insert was PCR'd from the MG1655 strain of E.coli K12.
MC31-rbs-luxI-Ter-Pcons2-rbs-luxR-Ter-Prlux-rbs-OMPA-golB-rbs-luxI-Ter It is a test of our positive feedback circuit for microcystin.
MC31, luxI, Pcons2, luxR, Prlux, OMPA-golB, Ter
The part codes for the human Oct-4 promoter, which regulates expression of the Oct-4 gene. The Oct-4 gene is essential for pluripotency in cells and is downregulated once differentiation occurs. Sequence information is from GenBank (NM_002701) and shows the cis-regulatory mRNA that controls transcriptional activation of the human Oct-4 promoter.
Wild-type found in T3 bacteriophage. Synthesized by IDT.
Promoter Family Member (U073O11+D038O22)
This RBS is part of the bacteriophage M13 genome, initiating translation of the gene IX message (BBa_M13009). The part aligns with base pairs 1190-1205 in M13K07 genome. It was identified as the RBS for gene IX message by Wezenbeek et al in Gene (1980) 11: 129-148
Upon recognition of a small molecule (e.g. nutrients or toxins), chemoreceptors transduce a signal through a set of Che proteins. This group of methylesterases and phosphatases regulates the rotation of the flagella. At the end of the chain of Che proteins is CheY. In phosphorylated state, CheY binds the flagella and causes the cells to tumble. The phosphatase CheZ dephosphorylates CheY, inducing dissociation of CheY from the flagella enabling the cells to swim. Cells lacking the CheZ protein are unable to swim and will tumble excessively and incessantly.
1 bp in BBa_K629003 was adapted to create an original restriciton site in the plasmid. CheZ was fused to GFP (BBa_K082003) and its LVA tag was codon-optimized to avoid introducing artificial DNA repetitions.
DNA sequences originate from the E. Coli genome and previous biobrick sequences.
Modified lambda P(RM) promoter: -10 region from P(L) and cooperatively repressed by 434 cI
Lamdba P(RM) promoter modified to be activated by lamda repressor (cI) and repressed by 434 repressor (cI). Two operator sites are used for both activation and repression to enhance cooperativity. Additionally, the -10 region from lambda P(L) was used instead of that from P(RM).
The O-R3 site of lambda (17 nt) was replaced by the O-R1 site of 434 (14 nt) to facilitate repression by 434 cI. The new 434 O-R1 is 5'-justified with the lambda O-R3 it replaced, leaving the -10 sequence intact. The 434 O-R2 site replaces the wildtype labmda promoter starting at -5, thereby preserving the 8 nt between 434 O-R1 and O-R2 in the wildtype. The mRNA transcript will contain an additional 9 nt compared to wildtype lambda phage. This part differs from BBa_I12036 because the -10 region from P(L) was used instead of that from P(RM).
Bushman, F. D. The bacteriophage 434 right operator roles of O-R1, O-R2, and O-R3. J. Mol. Biol. (1993) 230, 28-40. ; Lutz, R & Bujard, H. "Independent and tight regulation of transcriptional units in Escherichia coli via the lacR/O, the TetR/O and AraC/I1-I2 regulatory elements, Nucleic Acids Research, 1997, Vol.25, No.6 (1203-1210)
Zeocoin and bleomycin resistance cassette including promoter and coding sequence. It lacks a terminator.
Codon optimized for E coli by GenScript.
Streptoalloteichus hindustanus, PDB: 1BYL
This is the same sequence as STE2 promotor(BBa_K110008), but with suffix and prefix switched
This is the first part of the split Firefly luciferase. To be used with fLuc[2].
Promoter Family Member (U097NUL+D062NUL)
P_template1 serves as the truss for PCR cloning. By respectively applying 7 different primers upstream and downstream, that is, 49 pairs of primers in total, we have got 49 different promoters. Then we made them digsted and loaded into a vector that has already possessed a lacZ-alpha fragment. In this way, we can read from the color of the clones on the plate whether the inserted promoter works or not. By this means, We can try seven different positions for Operator A and seven for Operator B, thus constructing 7*7 kinds of promoters which have actually constituted a specific promoter family. After transformed into the four test plasmids (pCR00, pCR01, pCR10, pCR11) together with a downstream double-reporter system, we will get to know the solo-repression and co-repression effects of the two repressors on the specific promoter.
Regulated by the presence of Iron and UV light irradiation
GsiB ribosome binding site (RBS) for B. subtilis
GsiB is an endogenous ribosome binding site from ''B.subtilis''. The sequence of the gsiB ribosome binding site is '''AAAGGAGG''' which is complementary to the sequence '''UUUCCUCC''' from the 3' region of the 16s rRNA from ''B.subtilis''. GsiB is an endogenous ribosome binding site (RBS) from ''B.subtilis''. The sequence of the gsiB ribosome binding site is '''AAAGGAGG''' which is complementary to the sequence '''UUUCCUCC''' from the 3' region of the 16s rRNA from ''B.subtilis''. Previous research showed that the predicted binding energy of the 16s rRNA to the RBS is -9.3kcal.
Promoter Family Member (U085O11+D002O22)
Mid-expression level constitutive promoter in yeast
NOT Gate Promoter Family Member (D001O11)
TPP Down-regulated Hammerhead Ribozyme 2.5 with Native RBS+E0040+B0015
Yangyang ZHAO, Yan GONG, Zhenrun ZHANG, Qingyang XIAO
This part is TPP up-regulated hammerhead ribozyme, consisting of 131 nucleotides.
This part contains a native ribosomal binding site
This part was an engineered RNA controller composed of a TPP aptamer from B.subtilis and a hammerhead motif.
This RBS is part of the bacteriophage M13 genome, initiating translation of the gene V message (BBa_M13005). The part aligns with base pairs 827-842 in M13K07 genome. It was identified as the RBS for the gene V message by Wezenbeek et al in Gene (1980) 11: 129-148
Modified lamdba Prm promoter (cooperative repression by 434 cI)
Lamdba Prm promoter modified to be activated by lamda repressor (cI) and repressed by 434 repressor (cI). Two operators are used for both activation and repression to enhance cooperativity.
The O-R3 site of lambda (17 nt) was replaced by the O-R1 site of 434 (14 nt) to facilitate repression by 434 cI. The new 434 O-R1 is 5'-justified with the lambda O-R3 it replaced, leaving the -10 sequence intact. The 434 O-R2 site replaces the wildtype labmda promoter starting at -5, thereby preserving the 8 nt between 434 O-R1 and O-R2 in the wildtype. The mRNA transcript will contain an additional 9 nt compared to wildtype lambda phage.
Bushman, F. D. The bacteriophage 434 right operator roles of O-R1, O-R2, and O-R3. J. Mol. Biol. (1993) 230, 28-40.
PLRV Coat Protein gene - Isolated from Marfona potato
This part encodes the major coat protein of the Potato Leafroll Virus (PLRV). It was isolated from a PLRV infected Marfora potato leaf by iGEM team Wageningen UR 2012. This was done by cDNA synthesis followed by PCR. The PLRV major Coat Protein should be capable of forming a Virus-Like Particle, but this was not (yet) confirmed for this part.
PLRV-infected Marfora potato leaf (RNA).
PenP operator from Bacillus Lichenformis. Two operator sites are negatively regulated by PenI (C0074).
Promoter of flhB gene from E. coli flagella
''flhB'' is a class 2 gene that is part of the construction of the flagella ( ''Ordering Genes in a Flagella Pathway by Analysis of Expression Kinetics from Living Bacteria'' - S. Kalir, J. McClure, K. Pabbaraju, C. Southward, M. Ronen, S. Leibler, M. G. Surette, U. Alon ). Its promoter is consitutively repressed. It is activated by FlhD4C2 and maybe by FliA with different strength ( ''Using a Quantitative Blueprint to Reprogram the Dynamics of the Flagella Gene Network'' - Shiraz Kalir and Uri Alon ). '''Work in progress :''' Deeper quantitative characterization
The primers used to amplify this sequence are : *Forward : GTTTCTTCGAATTCGCGGCCGCTTCTAGAGCCACGTCATATCAGGCGGTCTGATAAGG *Reverse : GTTTCTTCCTGCAGCGGCCGCTACTAGTAGTTTTGTCGTCGCTCTCGTCAGACACGTC
Promoter Family Member with Hybrid Operator (D001O46)
The fhuA promoter. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
Construction of <PfhuA> basic part K112708 PCR of dv019/dv020 on MG1655 (225bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+225, L) Product is pBca1256-K112708 ----------------------------------------------- dv019 Forward biobricking of <PfhuA> cgataGAATTCatgAGATCTgctaagcgtgaaataccggatgg dv020 Reverse biobricking of <PfhuA> cgttaGGATCCactctgatgtaaagtgaatgataacg
Terminator to be placed upstream of multiple cloning site in new pSB series plasmids.
Three complete c-di-GMP riboswitches (Bc3, Bc4 and Bc5 RNA) with similar structures, which are arranged in tandem to constitute a triple-tandem (Bc3-5 RNA) riboswitch in the 5′-UTR of the cspABCDE mRNA in Bacillus thuringiensis subsp. chinensis CT-43.
We designed the sequence of bc3-5 by add perfix and suffix which are belong to BioBrick RFC 10.
From Bacillus thuringiensis subsp. chinensis CT-43 In the CT-43 genome19,20 (GenBank accession: NC_017208.1), five type I c-di-GMP riboswitches named Bc1, Bc2, Bc3, Bc4 and Bc5 RNA (Supplementary Tables S1???S5) were annotated in the Rfam database (http://rfam.sanger.ac.uk/search). Zhou, H., et al. (2016). "Characterization of a natural triple-tandem c-di-GMP riboswitch and application of the riboswitch-based dual-fluorescence reporter." Sci Rep 6: 20871.
This part is composed of an arabinose-inducible promoter for superfolded GFP attached by a linker to CpxR (on the C terminus of CpxR).
Constitutive Promoter, High Transcription
P2 promoter in agr operon from S. aureus
The agr P2 operon is an autocatalytic sensory transduction system in Staphylococcus aureus. P2 promoter regulates the synthesis of AgrB (a transmembrane protein) and AgrD (precursor of AIP autoinducing peptide). AIP binds to AgrC, a membrane binding receptor and it phosphorylates AgrA. The phosphorylated AgrA increases the expression level from P2 promoter.
Codon usage has been optimised for E. coli (which also seems to give good results for B. subtilis
The section of the sequence constituting the P2 promoter is subject to rather more debate than the sequences of the coding regions. [http://jb.asm.org/cgi/content/full/186/22/7549 Koenig, Robbin L., Ray, Jessica L., Maleki, Soheila J., Smeltzer, Mark S., Hurlburt, Barry K. Staphylococcus aureus AgrA Binding to the RNAIII-agr Regulatory Region J. Bacteriol. 2004 186: 7549-7555] shows the areas of the sequence to which phosphorylated AgrA binds while L Rao, R K Karls, and M J Betley: In vitro transcription of pathogenesis-related genes by purified RNA polymerase from Staphylococcus aureus. Bacteriol. 1995 May; 177(10): 2609???2614. shows the transcription start sites; we took the overall extent of the promoter region from that labelled in Morfeldt, E., K. Tegmark, and S. Arvidson. 1996. Transcriptional control of the agr-dependent virulence gene regulator, RNAIII, in Staphylococcus aureus. Mol. Microbiol. 21:1227???1237. (not available online).
Our terminator is based on strong, Rho-independent terminator of gyrase gene, which is type II topoisomerase (EC 5.99.1.3). DNA gyrase uses the energy of ATP hydrolysis to introduce negative supercoils or relaxes positive supercoils. Negative DNA supercoiling is essential in vivo to compact the genome, relieve torsional strain during replication, and promote local melting for vital processes such as transcript initation by RNA polymerase. Gyrase gene is an housekeeping gene and because of that it must have efficient terminator.
Terminator of gyrase (EC 5.99.1.3) gene.
Promoter Family Member with Hybrid Operator (D001O12)
This is a adenylate cyclase promoter that has been reported to oscillate in cell cycle dependant manner.
Ref: J. Biol. Chem., Vol. 260, Issue 5, 3063-3070, 03, 1985 Transcription of the Escherichia coli adenylate cyclase gene is negatively regulated by cAMP-cAMP receptor protein H Aiba [bases -53 to 12]
This part is the natural RBS associated with the invJ protein in Salmonella Typhimurium LT2.
This part was not designed with compliance to biobrick standard assembly in mind.
Ptet-rbs-rfp-Ter-Plac-rbs-tetR-Ter-Pcons2-rbs-lacI-Ter. It produces rfp when there is less lactose.
Ptet, rfp, Plac, tetR, Pcons2, lacI, Ter
mCYC promoter plus Zif268-HIV binding sites
This is a composite part containing the minimal promoter mCYC preceded by multiple consecutive binding sites for the Zif268-HIV binding domain. It contains the sequences of parts BBa_K165011 and BBa_K165016 but also contains additional DNA.
It was necessary to design primers for use in PCR to remove this part from its host plasmid and transfer it to a standard Biobrick vector.
Composite part that was taken via PCR from a synthetic plasmid containing the sequence. The plasmid came from Lawrence Berkeley National Laboratory.
Promoter Family Member (U097NUL+D002O22)
A Double Reporter (LacZ-alpha and GFP-AAV) loaded on pSB3C5. For more information please refer to BBa_I732401.
This provides the entire chloramphenicol resistance operon and will express CmR.
Game of Life - Secondary plasmid [tac pR, LuxI generator]
iGEM2009 Team:Southampton Game of Life secondary plasmid.
Regulated expression of LuxI (C0161) by TAC pR (K180000), a functional hybrid lac/trp promoter.
See relevant basic biobrick parts (K180000, B0034, C0161, B0015).
This is a ''vir'' promotor from ''Agrobacterium tumefaciens'' C58. It is located on the TI-plasmid before the ''virB'' genes. It is activated by phosphorylated ''virG'' response regulator.
The spv promoter. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
Construction of <Pspv> basic part K112707 PCR of dv017/dv016 on pBca1037 (499bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+499, L) Product is pBca1256-K112707 ----------------------------------------------- dv017 Forward biobricking of <Pspv> cgataGAATTCatgAGATCTaGATCCTGTGATGTTTGGCG dv016 Reverse biobricking of <Pspv> cgttaGGATCCGATAATGTtTGCAGGGGAATTATTTTG
Promoter 43 a constitutive promoter for B. subtilis
Promoter 43 is a constitutive promoter that constitutively expresses the P43 protein in ''B.subtilis''. This promoter has been shown to be recognized and active during the exponential and lag phases of growth. It has been hypothesized that the ability to recognize the promoter in exponential and lag phase of growth is due to the recognition of the promoter by both sigma factor 55 (the major sigma factor) and sigma factor 37 (the lag phase sigma factor) 1. The P43 promoter has been previously used for constitutive expression of exogenous genes within ''B.subtilis'' vectors 2. The context with which we used the promoter P43 is as a '''Polymerase Per Second''' (PoPS) generator.
The biobrick part was designed to include the binding sites for both the sigma factor A and B. In addition the biobrick standard was applied to the promoter P43 sequence.
The part was designed using the sequence from the ''B.subtilis'' genome and from previously published papers 2. This sequence was then synthesised by Geneart.
This is a modified version of the LacI promoter that should bind LacI tighter than the original.
This promoter is a modified version of the LacI promoter
This part is part of a family of Bioscaffold - Linkers. This part will help users interested in protein-fusion. The usage of the part will allow in frame assembly of 2(+) proteins by: *Removing the stop codons from the upstream protein. *Removing the scar created by the Assembly Standard 10 (RFC10) construction of 2(+) protein coding sequences. *Maintaining the start codon of the downstream protein. *Adding a flexible (Gly-Ser-Gly) linker between the two protein coding sequences. *Acting as a translational buffer if ligation is incorrect. ===Assembly Instructions=== *Create an Assembly Standard 10 fusion as below ; **Upstream Protein Part - Bioscaffold Linker - Downstream Protein Part** *Use the BioScaffold Specific Enzymes (i)Restrict with BpuEI to remove upstream scar (created between Upstream Protein - Bioscaffold) and stop codons from upstream protein part. (ii)Ligate and you will get the intermediate product; **Upstream Protein (w/out Stop codons) - Tyr - BioScaffold Linker - Downstream Protein** *Restrict with CspCI to remove downstream scar. *Ligate to get the final product; **Upstream Protein - Tyr-Gly-Ser-Gly- Downstream Protein** ====N.B.==== CspCI cuts with a 1 b.p discrepancy depending on the sequence. However this should not affect the restriction/ligation process as this has been taken into account and incorporated into the design. Even with the discrepancy involved the restriction/ligation process will still give the same product and in-frame fusion. The other restriction variants of CspCI have also been taken into account and relevant BioScaffolds are being produced to be tested. This version is for when CspCI cuts 13/10b.p. (top/bottom) on the 5' of the recognition sequence and 10/13bp (top/bottom) on the 3' of the recognition sequence.
The CspCI discrepancy had to be taken into account
Yeast Optimized sfGFP-N + ADH1 terminator + Kanamycin resistance
promoter to xylA without CRP and several binding sites for xylR
removed crp binding site removed rbs removed several xylR binding sites
This is the second part of the split Firefly luciferase. To be used with fLuc[1].
b0034 derived RBS to match spacing of J01122
RBS spacing modification to [RBS] such that the spacing between the shine-delgarno and the ORF is the same as it is in J01122.
This is the T3 promoter with an RBS site added. Other groups should use this part to regulate their part by adding it in front.
The T3 promoter comes from the T3 bacteriophage. The RBS site comes from...
This is the tightly regulated lac operon K389050 with a luciferase gene as readout.
lacIq integrated in promoter so good regulation, luciferase as sensitive readout
Promoter Family Member (U061O11+D062NUL)
A Double Reporter (LacZ-alpha and GFP-AAV) loaded on pSB3K5. For more information please refer to BBa_I732401.
Pm is the middle promoter of the Mu bacteriophage. It is in charge of the transcription of the activator protein C. Pm is activated by a product of the early transcription, protein Mor. It can be used to build a sensitivity tuner alongside this protein, this construct can be used to increase the amount of POPS in a certain genetic construction.
Synthetized from the reported sequence of Pm promoter from Mu bacteriophage.
Native ribosome binding site of sam8 (tyrosine ammonia lyase) from the bacterium Saccharothrix espanaensis DSM 44229.
Virtual part to be added to coding sequence I742142.
Allows for regulation of following gene with laqIq and IPTG...laqIq in same plasmid represses the promoter and IPTG proportionally depresses the promoter (Boer, et al., 1983).
Derived from trp and lac promoter sequences.
Promoter Family Member (U037O11+D002O22)
Weak1 RBS based on Ron Weiss thesis. Strength is considered relative to BBa_B0030, BBa_B0031, BBa_B0033.
Zif268-HIV binding sites + MET25 constitutive yeast promoter
Sequence comes from the natural B0030. Using B0030 as a medium strength RBS is from initial results from experiments characterizing all natural T7 RBSs.
The pR regulatory region is a 98 base-pair sequence with standard BioBrick prefix and suffix sections on its ends. It contains two operator sites at which HK022 cI protein can bind cooperatively. The -35 and -10 regions from pR are included along with some flanking sequences. The cutoff for the 5' end of the regulatory region was placed just after the pM -10 region which promotes transcription of a gene upstream (thus the pM -10 region is not included). The cutoff for the 3' end of the regulatory region was placed immediately prior to the RBS.
Derived from STHK022N . Literature claims that there is no cross-talk between HK022 cI regulatory region, Lambda cI, 434 cI and P22 cI, but this has not been verified experimentally by the synthetic biology group at MIT.
Double Promoter (cI, negative / TetR, negative)
promoter of Alcaligenes eutrophus PhaR gene
PCR using primers with biobrick prefix and surfix, cloned into pSB1AC3, and then eliminate Pst1 using site-directed mutation.
This is the Psid promoter taken from the P4 phage genome. It is an inducible promoter that is activated by a class of activators, including P2 ogr (I746350), PSP3 pag (I746351) and phiR73 delta (I746352). These different activators should cause different levels of activity of the Psid promoter.
J23105 plus B0034 is a regulatory part that can be used for both efficient transcription and translation. Just add any functional protein you want after it.
J23105 comes from constitutive promoter family from a small combinatorial library, B0034 is an RBS based on Elowitz repressilator.
The Ptac promoter is a functional hybrid promoter, derived from the trp and lac promoters, that are regulated by trp and lac [1]. This part also exist together with lacI, part BBa_K180000 [1] Proc. Natl. Acad. Sci. USA, Vol. 80, pp. 21-25
Oligos ordered from SigmaAldrich were annealed to form a double stranded DNA segment, ready to be ligated into any BioBrick backbone.
Created from oligos ordered from SigmaAldrich. Oligo sequences: Ptac+ AATTCGCGGCCGCTTCTAGAGGAGCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTA Ptac- CTAGTAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGATGATTAATTGTCAACAGCTCCTCTAGAAGCGGCCGCG
3' UTR site of alcohol oxidase 1 gene (aox1)
3' UTR site of alcohol oxidase 1 gene (AOX1) from K. pastoris used as a recombination site.
The DNA sequence from plasmid pPIC9K of Invitrogen (http://products.invitrogen.com/ivgn/product/V17520?ICID==%3Dsearch-product) was used for primer design. But the genomic DNA of Pichia pastoris wildtype X33 was used for amplification via PCR.
The recA(SOS)promoter gene from the chromosomal DNA isolated from MG1655 has been PCR amplified and inserted into plasmid pSB1A2(part number BBa_B0015).
The p22 cII regulatory region sequence is a 97 base-pair sequence with the standard BioBrick prefix and suffix sections on its ends. p22 cII repressor protein, BBa_C0053, binds to it. This segment contains O-R1, O-R2, a fragment of O-R3, the -35 of P-RM, and P-R (-10 and -35 from Tom Knight)
Regulatory region spanning 680 bp upstream of the start codon of the GPD1 gene in yeast. Very strong constitutive promoter.
Transcription terminator for the E.coli RNA polymerase.
Suggested by Sri Kosuri and Drew Endy as a high efficiency terminator. The 5' end cutoff was placed immediately after the TAA stop codon and the 3' end cutoff was placed just prior to the RBS of Gene 1.4 (before AAGGAG). Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator.
Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 V01146 .
Promoter Family Member (U049O11+D062O22)
T3-motni, terminator for Aspergillus niger
T3-motni is a terminator for use in Aspergillus niger.
Promoter Family Member with Hybrid Operator (D001O24)
Artificial terminator, estimated %T~<10% 8bp stem, 6nt loop Bidirectional, estimated reverse %T~=40%
Bidirectional, with the reverse estimated to be more effective than the forward. Has a polyA tail of 3 residues.
modified E. coli thr terminator, replaced all A-T pairs in stem with C-G pairs
Promoter Family Member (U097O11+D062O22)
Promoter of the copA copper efflux gene from Escherichia coli K12. This promoter is induced in the presence of copper ions, regulated by the activator protein CueR (CopR).
This version of the T7 0.3 RBS is designed for construction via Heather Keller's alternative assembly plan using XbaI and SapI for RBS/CDS assembly without a mixed site. Contains the -20 to -1 region of gp0.4 from the wild-type T7 genome. Can also be used with Standard BioBricks assembly, although this will add a 7 amino acid fusion to the N-terminus of the resulting protein.
This RBS is designed to be compatible with Heather Keller's RBS assembly scheme for creating RBS/CDS junctions without a mixed site. For use in this scheme, this part should be cut with XbaI and SapI. It must be used with a coding sequence containing the SapI site at the 5' end. This part is not compatible with CDSs that use a GTG site and must be used with CDSs using an ATG start site. The two bp spacer on the 3' end also allows this part to be used in standard biobricks assembly and is necessary to maintain the reading frame of a downstream CDS. Note that Standard Biobricks Assembly with this part results in the creation of a new ATG start site, and a 7 amino acid fusion at the N terminus of the downstream protein. CDSs with alternative start sites are compatible with this part via Standard Assembly. This part was inadvertently constructed without the single base pair (G/C) that separates the XbaI site and the 5' end of the part.
The RBS is taken from the wild-type bacteriophage T7 genome. Please see part B2001 for more information.
Promoter of flhDC gene from E. coli flagella
''flhDC'' is the only class 1 gene that is part of the construction of the flagella. It is the master regulator of the biosynthesis of the flagella ( ''Ordering Genes in a Flagella Pathway by Analysis of Expression Kinetics from Living Bacteria'' - S. Kalir, J. McClure, K. Pabbaraju, C. Southward, M. Ronen, S. Leibler, M. G. Surette, U. Alon ). The promoter of ''flhDC'' is regulated by many transcription factors, for instance, OmpR* and EnvZ* are two repressors of this gene. '''Work in progress :''' Characterization of the repression by OmpR* and EnvZ*
The PCR on colony to amplify the promoter of ''flhDC'' did not work. We amplified both the promoter and the gene and then we did a specific amplification of the promoter from the full operon. We had a PCR amplicon of the right length this time. The primers used to amplify the promoter and the gene are : * Forward : GTTTCTTCGAATTCGCGGCCGCTTCTAGAGTTGTATGTGCGTGTAGTGACGAGTACAG * Reverse : GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTAAACAGCCTGTACTCTCTGTTCATCC Then to amplify only the promoter we used : * Forward : GTTTCTTCGAATTCGCGGCCGCTTCTAGAGTTGTATGTGCGTGTAGTGACGAGTACAG * Reverse : GTTTCTTCCTGCAGCGGCCGCTACTAGTATCCCACCCAGAATAACCAACTTTAT
The spv2 promoter. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
Construction of <Pspv2> basic part K112706 PCR of dv015/dv016 on pBca1037 (500bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+ 500, L) Product is pBca1256-K112706 ----------------------------------------------- dv015 Forward biobricking of <Pspv2> cgataGAATTCatgAGATCTccttatgcagcgtaagggccgcaac dv016 Reverse biobricking of <Pspv2> cgttaGGATCCGATAATGTtTGCAGGGGAATTATTTTG
NOT Gate Promoter Family Member (D001O66)
Assambly of [http://parts.igem.org/Part:BBa_J23116 J23116] and lacI-binding-site of [http://parts.igem.org/Part:BBa_R0010 R_0010] The "lacI binding site" of Part [http://parts.igem.org/Part:BBa_R0010 R_0010] differs from lacO from Part [http://parts.igem.org/Part:BBa_J33207 BBa_J33207].
Double terminator + Kanamycin resistance cassette + Double ter
This part contains a double terminator and a functional kanamycin resistance cassette (promoter, resistance against kanamycin and a double terminator, BBa_B0014 + BBa_P1003 + BBa_B0014). It is reliable to design a recombinant vector by gene insertion in a non lytic bacteriophage as the Lambda phage. In fact it allows us to know if a bacteriophage is modified or not and if the inserted gene is expressed or not. When the virus is modified the kanamycin cassette induces a kanamycin resistance, so the bacteria become resistance, and when the virus is not modified bacteria are killed by kanamycin. We can use this part as a reporter, or as a simple resistance cassette. The double terminator allows the RNA translation arrest before the resistance cassette, so this is already created to insert a coding gene with its RBS before the resistance cassette.
When we design the bio-brick it was impossible to obtain a functional bio-brick by using the plasmid SB1AK3. But it's worked with a pSB1A1.
Part:BBa_B0014, Designed by Reshma Shetty Group: Registry (2003-07-16) Part:BBa_P1003, Designed by Reshma Shetty Group: Knight Lab, MIT (2006-01-23)
The kozak sequence acts as a eukaryotic RBS. It is cloned directly between a promoter and coding region.
This is a pRecA promoter with LexA binding sites. LexA is cleaved by RecA when RecA is upregulated due to induction of the SOS response triggered by single stranded DNA. For this part to work it must be in a RecA+ strain of E. coli such as TG1
How far past the start site of transcription do you need to go in order for the promoter to drive transcription? The promoter has been confirmed to drive transcription in a regular DH5alpha strain, so it the answer to the question is, 3 bp before the suffix, maybe less. The LexA binding site is in the core region of the promoter (between the -35 and -10 sigma 70 binding sites). According to Robert Sidney Cox III and colleagues in their paper "Programming ene expression with combinatorial promoters, the core region is the most effective region for binding of repressor molecules.
synthetic. Oligonucleotides were designed to form the double stranded molecule with sticky ends for direct ligation into a biobrick vector.
T2-motni, terminator for Aspergillus niger
T2-motni is a terminator for use in Aspergillus niger
THIS PART DOES NOT WORK. Need to add the CI operator sites.
A inducible promoter plus a strong RBS B0034, repressed by TetR, induced by tetracycline and aTc.
This part should be used along with TetR.
B0034 is based on Elowitz repressilator.
Assambly of [http://parts.igem.org/Part:BBa_J23119 J23119] and lacI-binding-site of [http://parts.igem.org/Part:BBa_R0010 R_0010] The "lacI binding site" of Part [http://parts.igem.org/Part:BBa_R0010 R_0010] differs from lacO from Part [http://parts.igem.org/Part:BBa_J33207 BBa_J33207].
Promoter Family Member (U061O11+D014O22)
The Gli1 and LexA transcription factor binding sites on the mCYC minimal promoter (in that order.) Transcription regulation comes from Gli1 and LexA-binding transcription factors.
Assambly of [http://parts.igem.org/Part:BBa_J23118 J23118] and lacI-binding-site of [http://parts.igem.org/Part:BBa_R0010 R_0010] The "lacI binding site" of Part [http://parts.igem.org/Part:BBa_R0010 R_0010] differs from lacO from Part [http://parts.igem.org/Part:BBa_J33207 BBa_J33207].
V.cholerae ctx gene promoter region. Transcription takes place In the presence of a ToxR dimer. We don't have the -10 and -35 sites annotated (need to do this).
GENBANK M21250
An intermediate part designed to be used with the AND gate designed by Caltech iGEM '10: [http://2010.igem.org/Team:Caltech/Project Project Details].
Designed to be placed upstream of positive feedback mechanisms and desired output genes.
Promoter Family Member (U061O11+D050O22)
Promoter Family Member (U097O11+D062NUL)
Artificial terminator, estimated %T~>90% *8bp stem, 6nt loop *Bidirectional, estimated reverse %T~>90%
Bidirectional, with the reverse estimated to be less effective than the forward. Has a polyA tail of 9 residues.
This part has a fused protein downstream of an arabinose-inducible promoter: CpxR-IFP[1]. Biobrick BBa_K1486013 has CpxR-IFP[2] with an arabinose promoter. This construct was made in both orientations: BBa_K1486014 is IFP[1]-CpxR (with an arabinose promoter).
Both orientations were made, as well as for IFP[2] so all dimerizations could be tested (see main page for biobrick numbers). Flexible linkers were used to fuse both proteins, and an RBS was put before each one of them.
This pLas' promoter contains the regulatory region upstream of the LasI gene in Pseudomonas aeruginosa. K091117 includes a LasR binding site as well as a -10 region modified from CATAAA to TATAAA to reflect our pLasXOR promoter design. Activation is expected to occur in the presence of the autoinducer PAI-1 and the LasR protein. The pLas' promoter serves as a control for the evaluation of the pLasXOR promoter, part BBa_K091118.
To minimize leaky transcription, a secondary transcriptional start site was excluded from the design.
The sequence of the LasI upstream regulatory region is available at http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=176640
Positively regulated, OmpR-controlled promoter. This promoter is derived from the upstream region of ompC. Phosphorylated OmpR binds to the operator site and activates transcription. This is a truncated version of BBa_R0082.
The promoter deletes the C2 and C3 regions of BBa_R0082 (bases 37-74 in BBa_R0082) and inserts an 8-base linker before the -35 and -10 signal region. The efficacy of this promoter versus the full ompC upstream region (BBa_R0082) or the ompF upstream region (BBa_R0084) is currently unknown. The a at base 41 is mutated to t to avoid XbaI site. Speculation that this was a previous assembly site.
Complete sMMO of Methylococcus capsulatus
The part consists of the six subunits of methane monooxygenase (MMO). Gene order: mmoX, mmoY, mmoB, mmoZ, mmoD, mmoC The expression is under control of lac+pl. MMO converts methane to methanol.
Part is designed considering the RFC10 standard.
Promoter Family Member (U049O11+D026O22)
This part contains both fragments of the split renilla luciferase, each of them preceded by an Elowitz RBS.
trpC terminator for Aspergillus nidulans
TtrpC is a terminator of tryptophan synthase of Aspergillus nidulans.
From the plasmid p71 from the Center for Microbial Biotechnology at the Technical University of Denmark plasmid collection.
Promoter Family Member (U097O11+D026O22)
pcI (for positive control of pcI-lux hybrid promoter)
Placement of the operator sites was optimized for steric interference between the C2003 and RNA polymerase based on structural models.
Promoter Family Member (U037O11+D038O22)
This part has two fused proteins downstream of an arabinose-inducible promoter: CpxR-IFP[1] and CpxR-IFP[2]. This construct was made in all orientations: - IFP[1]-CpxR + IFP[2]-CpxR (BBa_K1286009) - IFP[1]-CpxR + CpxR-IFP[2] (BBa_K1486010) - CpxR-IFP[1] + IFP[2]-CpxR (BBa_K1486011)
This part was inadvertently made while trying to make BBa_R2111. It contains a single base pair mutation in the -10 site of BBa_R2111.
T7 promoter with an A->T mutation at the -16 position of the consensus sequence
K091157 is a modified version of the pLux' promoter (BBa_K091156) containing the addition of a LasR binding site between the -35 and -10 regions. This promoter is expected to be activated in the presence of AI-1 and LuxR and repressed in the presence of PAI-1 and LasR.
In order to fit the LasR binding site between the -35 and -10 regions, the last base of the Las Box was cropped and the -35 region was modified to overlap with the first two bases of the Las Box.
The LasR binding site is from the region upstream of the LasI gene in Pseudomonas aeruginosa. The rest of the promoter is the same as BBa_K091156.
Promoter Family Member (U097O11+D038O22)
pspoIIE promoter (spo0A J01004, positive)
promoter from b. subtilis, activated by spo0A
Parts J23100 through J23119 are a family of constitutive promoter parts isolated from a small combinatorial library. B0030 is a strong RBS based on Ron Weiss thesis
optimized (TA) repeat constitutive promoter with 21 bp between -10 and -35 elements
This is the core of the CYC1 promoter of ''S. cerevisiae''. This promoter element allows basal transcription. The transcription signal can be modulated by adding operator sites upstream of this BioBrick. In that way activators or repressors can act on the transcription machinery. <br<
This BioBrick contains the TATA-box and most of the 5'UTR (-139 to -36, relative to the translational start)of the natural CYC1 promoter. A Kozak sequence is not included and has thus to be added!
position 5 mutated promoter lux pR-5 (luxR & HSL regulated)
The position 5 mutated promoter lux pR-5 is a product of site-directed mutagenesis of promoter lux pR (BBa_R0062). The promoter lux pR is based on the Vibrio fischeri quorum sensing gene promoters. The position 5 mutated promoter lux pR-5 gives a higher expression of downstream genes in an autoinduction system. In another word, the lux pR-5 has a higher efficiency than the lux pR. Similar to promoter lux pR, the expression is up-regulated by the action of LuxR protein combined with the signal molecule HSL.
By PCR method the G residue of position 5 was changed to a C based on the sequence of the promoter lux pR(BBa_R0062).
This is a modification of the standard plac operon with everything before the -35 site removed, the Or2 site from the lambda promoter inserted at the beginning, and a 17-bp spacer added between the two.
Hybrid promoter: HSL-LuxR activated, P22 C2 repressed
Hybrid promoter consisting of the Lux box which overlaps partly with -35 box. Binding sites for P22 C2 OR2 and OR1 are located between the -35 and -10 boxes and downstream of them respectively.
-35 box is partly composite. -10 is taken from the P22 phage PR regulatory region
Weaker version of wild type pBAD ( I13453 ).
Site-directed mutagenesis on I13453 with the following primers: Forward: TAATCTTATGGATAAAAAAGCTATGGCATAGC Reverse: GCGGATCCTACCTGACGCTTTTTATC
Specialized RBS modified from that of B0036 (Brink et al.)
This RBS has the modified Shine-Dalgarno sequence, it has a reduced secondary structure relative to using the actual Brink sequence with mixed connective sites flanking it. the spacing between the SD and the ATG is the same as that used by Brink et al. even though it is non-optimal for E. coli
osmY's promoter is active in stationary phase and under high osmotic pressure conditions.
Truncated Forward Primer: 5'- GTT TCT TCG AAT TCG CGG CCG CTT CTA GGCT TAT GTT TTC GCT GAT ATC - 3' Total Length: 49 bp Annealing Length: 21 bp GC Content: 38.1% Melting Temperature: 49.5 degrees C hairpin deltaG: -2.66 kcal/mol self dimer deltaG: -99.97 kcal/mol Reverse Primer: 5'-GTT TCT TCC TGC AGC GGC CGC TAC TAG TAT TGT TAA ATA TAG ATC ACA ATT TTG- 3' Total Length: 54 bp Annealing Length: 25 bp GC Content: 20.0% Melting Temperature: 46.4 degrees C hairpin deltaG: -2.86 kcal/mol self dimer deltaG: -99.44 kcal/mol heterodimer deltaG with Conserved Forward Primer: -99.97 kcal/mol
This part was PCRed out of E. coli's genome.
unidentified/uncharacterized CinR dependent
Constitutive Aspergillus nidulans promoter
Cloned into shipping plasmid pSB1C3 with USER tails
Aspergillus nidulans genomic DNA (AN1122)
Srini Devadas, David Gray, Ronny Krashinsky, Debra Lin, and Chris Zheng Liu
This part involves the -10 binding site, the -35 binding site, and the twenty nucleotides between that constitute the lux box. With this part, LuxR functions as a acyl-homoserine lactone-dependent repressor. LuxR resonds to the HSL produced by LuxI, N-(3-oxohexanoyl)-HSL. The Lux box is positioned such that it partially overlaps the consensus -35 and -10 hexamers of an RNA polymerase binding site.
This cold shock promoter is only active at temperatures lower than 30 degree Celsius.
This is a modified K112400 heat shock promoter in BBa standard.
{| border="1" | |align="center"| |align="center"| |}
ptrA, selection marker for Aspergillus nidulans
LasR/LasI Inducible & RHLR/RHLI repressible Promoter
This part is similar to BBa_R0079 but has an added binding site for RHLR/RHLI in between the -35 and -10 region. Thus this promoter is induced by LasR/LasI but repressed by RHLR/RHLI binding.
The natural spacer between the -35 and -10 is 14bp and the natural O1 site for RHLR/RHLI is 20bp long. There was not much over lap of the -35 or the -10 with this sequence, so it was nessicary to chop off some of the binding site sequence in order to maintain the correct spacing and preserve the -35 and -10 sequence. The binding site sequence is missing a tcc on is 5' end and a t on its 3' end.
Rust L. et. al., "Analysis of the Pseudomonas aeruginosa Elastase (lasB) Regulatory Region". Journal of Bacteriology, 1996.
Assambly of [http://parts.igem.org/Part:BBa_J23104 J23104] and lacI-binding-site of [http://parts.igem.org/Part:BBa_R0010 R_0010] The "lacI binding site" of Part [http://parts.igem.org/Part:BBa_R0010 R_0010] differs from lacO from Part [http://parts.igem.org/Part:BBa_J33207 BBa_J33207].
This is the promoter sequence of g3 which has been separated from g8. It also includes the restriction enzyme EcoRV in front.
This part used in conjunction with BBa_M131520 allows g8 and promoter of g3 to be separate from each other, therefore allowing free regulation of g3 with no consequences on g8.
ftsAZ promoter is a promoter with levels of expression are inversely proportional to the growth rate. This part is actually in BBb format. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR al0021/al0022 on MG1655 (804bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112320 ----------------------------------------------- al0021 Forward Biobricking of ftsAZ promoter CCATAGAATTCatgAGATCTaacgacagtgttggtgagcg al0022 Reverse Biobricking of ftsAZ promoter CGttaGGATCCgtaccaatctccagtcctactaccag
Lac and sigma 28 conserved binding sites
MC7-rbs-luxI-Ter-Pcons2-rbs-luxR-Ter-Prlux-rbs-OMPA-golB-rbs-luxI-Ter. It is a test of our positive feedback circuit for microcystin.
MC7, luxI, Pcons2, luxR, Prlux, OMPA-golB, Ter
Promoter activated by RhlR in concert with C4-HSL. C4-HSL is produced by RhlI, and acts as a quorum-sensing autoinducer. This is the natural sequence taken from Pseudomonas aeruginosa. Crosstalk: this promoter is also activated at a low level by LasR with its associated HSL.
The -10 and -35 sites are labeled as identified in [Pearson97]. The part begins with the RhlR binding site, and (rather arbitrarily) ends with the first transcribed base (+1).
Pseudomonas aeruginosa rhlAB promoter
Kanamycin resistance cassette and a double terminator
This part contains a kanamycin resistance cassette and a double terminator (BBa_P1003 + BBa_B0014). This allows the kanamycin cassette to be functional. The cassette can be used to design a recombinant vector by gene insertion in a non lytic bacteriophage as the Lambda phage. In fact it allows us to know if a bacteriophage is modified or not and if the inserted gene is expressed or not. When the virus is modified the kanamycin cassette induces a kanamycin resistance, so the bacteria become resistance, and when the virus is not modified bacteria are killed by kanamycin. This part is completed by B0014 + P1003 + B0014 to finish the transcription before the resistance cassette.
Part:BBa_P1003, Designed by Reshma Shetty Group: Knight Lab, MIT (2006-01-23) Part:BBa_B0014, Designed by Reshma Shetty Group: Registry (2003-07-16)
This is the hybB promoter, which normally controlls hydrogenase regulation at low temperatures. It turns ON at 30oC and is active as low as 20oC (lower not tested). It is OFF at 37 and higher temperatures. In the literature, it is also active in anaerobic environments. This hasn't been tested.
lizjaneclarke@gmail.com Univerity of California - San Francisco
This part contains the LexAoperator and MajorLatePromoter genes with restriction site KdeI at the 5 prime end and AscI at the 3' end. Will use it as both a regulatory part for certain signals as well as a promoter.
Had to make sure the restriction sites used were not in the sequence or in the sequence of any of the genes downstream of the promoter.
Promoter Family Member (U097NUL+D014O22)
A Double Reporter (LacZ-alpha and RFP) loaded on pSB3C5. For more information please refer to BBa_I732402.
rLucN + ADH1 terminator + Kanamycin resistance
This is the tightly regulated lac operon BBa_K389050 with a mRFP gene as readout.
lacIq integrated in promoter so good regulation, mRFP as readout
DNA sequences originate from the E. Coli genome and previous biobrick sequences. The ribosome binding site was chosen from the Anderson RBS family.
promoter of phoB in E.coli, an external phosphate regulated promoter which can be activated when encountering phosphate limitation.
The promoter region should contain PhoB binding site
Double terminator + constitutive promoter
This part contains a double terminator and a constitutive prokaryotic promoter. It is a simple promoter to begin gene transcription. For Sup???biotech-Paris (2009) team this part is used after to be added to a recombinant lambda phage to begin the transcription of synthetic genes inside the Lambda phage genome.
BBa_B0014 : Designed by Reshma Shetty Group: Registry (2003-07-16) BBa_J23100 : Designed by John Anderson Group: iGEM2006_Berkeley (2006-08-04)
The T0 transcriptional terminator terminates transcription with 80-90% efficiency.
Tais Sineiro Herig, Raissa Estrela Curado and Gleidson Silva Teixeira
Double Promoter (TetR, negative / P22 cII, negative)
This is the natural RBS from the sicA chaperone (BBa_J6021) in the Salmonella Type III Secretion System.
This part was not designed with standard biobrick assembly in mind.
This a GFP reporter regulated by a self-cleaving hammerhead ribozyme. The self-cleaving of the hammerhead ribozyme would lead to the formation of the intron P1 stem, thus triggering the self-splicing of the intron.
The self-cleaving of the hammerhead ribozyme would lead to the formation of the intron P1 stem, thus triggering the self-splicing of the intron.
The intron derives from the genome of Cd
The pRE coding region from λ phage. It is able to be induced by CII (BBa_K116602).
constitutive promoter with (TA)10 between -10 and -35 elements
With 10 TA repeats between the -10 and -35 elements, the distance between the two elements is optimal for the sigma factor to bind to the promoter. Thus, the promoter should be in the 'on' state.
Promoter Family Member (U037O11+D062NUL)
A Double Reporter (LacZ-alpha and GFP-AAV) loaded on pSB3K5. For more information please refer to BBa_I732403.
---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
Biobricking of cadC promoter and cadC gene PCR cadC-F and cadC-R on MG1655 (2336 bp, EcoR1/BamH1) Sub into pBca1256 (EcoR1/BamH1 910+2472bp, L) Product is pBca1256-K112406 ---------------------------- cb10013-cadC-F cgataGAATTCatgAGATCTcaccagcaatttgcgggcatatc cb10012-cad-R cgtagGGATCCtgaccagaccaaactggcg
Diguanylate cyclases produce cyclic diGMP (c-di-GMP), a secondary messenger. Diguanylate Cyclase (DgcZ), also called YdeH, is present in Escherichia coli and the stimulus that controls it is Zinc.
The sequence did not have any restriction sites to remove so it was immediately PCRed out of the genome and had the registry prefix and suffix added to it. It was then ligated into the pSB1C3 vector. YdeH however, is toxic to E. coli cells.
The source of this part is Escherichia coli MC40100 genomic DNA which was provided by the Tracy Palmer lab in the Molecular Microbiology department of the University of Dundee, UK.
Promoter Family Member (U073O11+D014O22)
PompR is a OmpR-controlled promoter which can be positively regulated by phosphorylated OmpR. This promoter is taken from the upstream region of ompC. Phosphorylated OmpR binds to the three operator sites and activates transcription. We insert PompR(BBa_R0082) sequences into the upstream region of rfp gene, which together is connected to vector PSB1C3 afterwards, and therefore we are able to observe the OmpR promoter efficiency through the control of light.
We constructed this biobrick through pcr.
This is the promoter for the ''Eschaerichia coli'' JM109 ''cstA'' gene. It includes the CRP-binding site and the RNA polymerase-binding site. Low glucose concentration results in increased activity by adenylate cyclase. cAMP binds to the cAMP receptor protein, which, in its bound form, is able to associate with the promoter and promote transcription of the downstream gene. (''cstA'' encodes the carbon starvation protein.)
Constitutive promoter J23100 with Lac system inverter controlling the expression of LasR
The biobricks BBa_J23100 and BBa_K266003 where used for build this biobrick
This RBS is recognized by ribosomes carrying the modified anti-Shine Dalgarno sequence as found in Brink et al. (see below)
Brink MF, Verbeet MP, de Boer HA (1995). Specialized ribosomes: highly specific translation in vivo of a single targetted mRNA species. Gene, 156, pp. 215-222
Vinay S Mahajan, Brian Chow, Peter Carr, Voichita Marinescu and Alexander D. Wissner-Gross
The cI regulated promoter is based on the pR promtoer from bacteriohage lambda. The promoter has two two DNA binding sites for lambda cI repressor <bb_part>BBa_C0051</bb_part>. cI binding results in repression of transcription. The specific sequence used here is based on the cI repressible promoter used in the Elowitz repressilator (and references therein).
In order to address concerns about the promoter transcribing in the reverse direction, we have removed the -35 and -10 signals responsible for the promoter activity in the reverse direction. (More details needed here! DE, 2/24/03) Incompatible with host expressing cI repressor.
Inducible Green Fluorescent Protein UnaG+6xHis-tag
Natural RBS sequence for Bacillus Subtilis, described in the literature as a strong RBS for this bacteria.
From genomic sequence Bacillus Subtilis.
This is the -20 to -1 region of T7 gp0.7, which contains the native RBS region for this gene. The physical instantiation of this part does not exist. This part is not compatible with Standard Biobricks assembly methods, as the scar created during assembly would interrupt the spacing between the RBS and the start codon of the CDS. Individuals wishing to use this RBS sequence in their assembles should refer to part XXXXX. (Information forthcoming)
The -20 to -1 region was chosen to be representative of the RBS region of the T7 genes for the following reasons: 1) The Shine-Delgarno region is contained within this defined region for all T7 genes. 2) Current literature suggests that when bound to the Translation Start Site (0), the Ribosome covers positions -20 to +13.
Genome of wild-type bacteriophage T7, positions 2001-2020. Includes the -20 to -1 region of gp0.7.
bolA promoter is a promoter where levels of expression are inversely proportional to the growth rate. To read more, go to following link [http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=402084&blobtype=pdf] This part is actually in BBb format. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR al0017/al0018 on MG1655 (467bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112318 ----------------------------------------------- al0017 Forward Biobricking of bolA promoter CCATAGAATTCatgAGATCTtgctgtggcagtgtaatcgtc al0018 Reverse Biobricking of bolA promoter CGttaGGATCCcttctatccgctcacgtatc
This composite regulatory part can be repressed by LuxR and activated by LuxR-3OC6HSL. Induced Plux is a strong promoter and B0034 is a strong RBS, which makes it can be used for both transcription and translation.
Plux is from V. fischeri and B0034 is based on Elowitz repressilator.
This BioBrick is a tightly controlled ''lac'' operon. It contains a lacIq and a ''lac'' operator, so it is inducible with IPTG but has a low basal expression. It is also possible to use this BioBrick in strains that do not carry a lacIq like ''E. coli'' TOP10.
we needed a tightly controlled inducible promoter
Lux pI+RBS+LuxR+2TM+Lux pR+RBS+LuxI+2TM+Lux pR+RBS+GFP+2TM+Lux pR+RBS+Ndh+2TM
Ptet-rbs-rfp-Ter-Plac-rbs-tetR-Ter. It produces rfp when there is enough lacI or less lactose.
Xuenian chen, Zewei yan and Yichong wang
α-ALS gene express acetolactate synthetase that contribute to produce diacetyl. The synthetize pathway of diacetyl, in E.coli, beginning with the central pathway metabolite pyruvate as the precursor molecule, the first step in the diacetyl biosynthetic pathway involves the condensation of two molecules of pyruvate to yield one molecule of acetolactate, and is catalyzed by acetolactate synthase (ALS).The stability of acetolactate is known to be affected by the presence of oxygen, which causes its spontaneous decarboxylation to produce diacetyl.
Pdps promoter. This part is actually in BBb format. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR JH089F/JH089R on Salmonella L2. (379 bp, EcoRI/BamHI/DpnI) Sub into pBjh1601CK (EcoRI/BamHI, 3134+910, L) Product is pBjh1601CK-K112322 ----------------------------------------------- JH089F Forward biobricking of {Pdps} cgataGAATTCatgAGATCTaacgattcgacgcgaaccgc JH089R Reverse biobricking of {Pdps} cgttaGGATCCaatctcatatcctcttgatg
Constitutive Promoter, Medium Transcription
Right facing promoter (for xylA) controlled by xylR and CRP-cAMP
removed RBS cut out several xylR activation sites
CRP-cAMP transcriptional dual regulator activates transcription
XylR-Xylose transcriptional activator activates transcription
Double Promoter (constitutive / TetR, negative)
rLucC + ADH1 terminator + CaUra3 cassette
Promoter Family Member (U037O11+D050O22)
Promoter Family Member (U085O11+D026O22)
T7 promoter with an T->G mutation at the -17 position of the consensus sequence
Hygromycin cassette for selection of mammalian cells. The selection cassette contains the SV40 promoter, the hygromycin resistance gene, and the SV40 poly A as terminator
Compatible with the Plu'n'Play assembly standard
From plasmid pcDNA3.1/Hygro from Invitrogen
Calgary's BBa_K339007 repaired (part didn't have the sequence they said they had.
A lac repressible T7 promoter. This promoter is similar to those found in the pET vectors with T7lac promoters. I mutated the -9 base to an A, which should make it less than 3% of the strength of consensus. The base numbering is as used by Studier and coworkers (JMB 1991, 219, 45-59). See BBa_R0183 for more information.
The -9 mutation from consensus was included to weaken the consensus promoter as described by Imburgio and co-workers (see BBa_R0183 for more information)
This sequence is based on the T7lac promoter described by Dubendorff and Studier (JMB 1991, 219, 45-59). The only modification is the mutation at -9.
Promoter, Zif23 regulated, test: between and after
Promoter with standard -10 and -35 sites. A symmetric Zif23 dimer target site is between the -10 and -35 sites, and a symmetric Zif23 dimer target site is after the -10 site.
Double terminator consisting of BBa_B0010 and BBa_B0012
B0015+pBAD+vioABD+B0015+pBAD+TPP Down-regulated Hammerhead Ribozyme 2.5+vioE
the Ribosomal Binding Site of vioE is contained in TPP Ribozyme.
Hammerhead Ribozyme backbone comes from E.coli, and TPP aptamer comes from B.subtilis.
This part is from the bacteriophage M13 genome, bp 381-428 in M13K07. It directs transcription of M13 gene X (BBa_M13510, BBa_M13010). Its identity as a promoter was described in Gene (1980) 11:129-148 based on -10,-35 homology and fragment of genome able to bind RNAP.
A regulatory part composed of a strong promoter and a strong RBS, which can be used in both transcription and translation. Just add any protein after it.
J23119 is from a constitutive promoter family and B0034 is based on Elowitz repressilator.
Promoter for grpE gene - Heat Shock and Ultrasound Sensitive
According to Vollmer, et. al in their 1998 Applied and Enviormental Microbiology paper, "Bacterial Stress Responses to 1-Megahertz Pulsed Ultrasound in the Presence of Microbubble", the promoter for the grpE gene, a known heat shock promoter, is upregulated 24 times when cells are exposed to high frequency ultrasound and microbubble cavitation.
Promoter Family Member (U073O11+D002O22)
Assambly of [http://parts.igem.org/Part:BBa_J23106 J23106] and lacI-binding-site of [http://parts.igem.org/Part:BBa_R0010 R_0010] The "lacI binding site" of Part [http://parts.igem.org/Part:BBa_R0010 R_0010] differs from lacO from Part [http://parts.igem.org/Part:BBa_J33207 BBa_J33207].
This is the natural RBS for the sptP gene in Salmonella Type III Secretion
This part was designed without consideration for biobrick standard assembly. This RBS has not been explicitly tested for expression rate.
Double Promoter (cI, negative / LacI, negative)
This is a composite part containing the minimal promoter mCYC preceded by multiple consecutive binding sites for the Gli1 binding domain. This part contains the sequences for BBa_K165012 and BBa_K165016 but also has some additional DNA.
The ribosomal binding site of the creD gene in the E Coli creD operon.
Promoter driving the lasI gene in Pseudomonas aeruginosa. Activated by LasR+3OC12HSL and not RhlR+C4HSL (Seed et al., 1995) ~4x stronger than the lasB promoter (BBa_R0079; Seed et al., 1995). Switch point ~0.1nM 3OC12HSL (Seed et al., 1995)
Promoter sequence cropped to 4nt upstream of Las box according to example of Whiteley and Greenberg, J. Bac, 2001
Promoter Family Member (U049O11+D038O22)
This part contains both fragments of the split firefly luciferase, each of them preceded by an Elowitz RBS.
fiu ??? Outer Membrane Ferric Iron Transporter
===Biology of Fiu=== The fiu gene encodes for an outer membrane transporter found in E.coli, a gram negative bacterium. It is involved in the active transport (energy dependent uptake) of ferric iron from the extracellular environment. This outer membrane receptor can only bind ferric iron and is a catecholate siderophore. For uptake into the cell to be completed fiu has to complex with another protein known as TonB (a transperiplasmic protein) that will carry out the active transport by using ATP as an energy source. ===What this Biobrick gives you upon expression=== This biobrick encodes ONLY for fiu. ===Fiu Statistics=== Fiu belongs to the family of Ton-B dependent receptors and is related to fhuA, hence it also has a beta-barrel structure. {| style="color:white; background-color:#0066CC;" cellpadding="20" cellspacing="0" border="2" !Chassis !Monomer/Multimer !Aminoacid Length/Weight !Localisation !Structure |- |E.Coli |Monomer |760/81.96kDa |Outer Membrane/OMV |beta-barrel |} ===References=== [http://ecoliwiki.net/colipedia/index.php/fiu:Gene_Product%28s%29#Domains.2FMotifs.2FModification_Sites E.Coli wiki net] Riley, M. et al. (2006) Nucleic Acids Res 34:1-6 (corrected supplemental data from B. Wanner) [http://www.ecogene.org/geneInfo.php?eg_id=EG13317 Ecogene number EG13317] [http://www.uniprot.org/uniprot/P75780 UniProt Accession number P75780]
In order to comply with assembly standard 10 (RFC10) the stop codon for fiu had to be changed to TAA.
Fiu gene is part of the chromosomal E.coli genome. In particular it is found at 18.07min with orientation in the counter-clockwise direction. [http://www.ecogene.org/geneInfo.php?eg_id=EG13317 Further details at EcoGene]
optimized (TA) repeat constitutive promoter with 17 bp between -10 and -35 elements
The MioC promoter is a cell-cycle dependent promoter that is repressed before initiation of replication and depressed shortly after. The regulation of the MioC gene is overseen by several DNAa proteins, which bind along the promoter. This particular MioC promoter was isolated from K-12 (JM109). This promoter can be used in series with a reporter to signal only during a given part of the cell cycle.
Site directed mutagenesis was used to create a Spe1 restriction site.
The source of this part is from the genomic dna of E.coli (JM109).
Gram-Positive Strong Constitutive Promoter
This is a strong constitutive (always-on) promoter for use with gram-positive organisms. It requires a downstream gram-positive ribosomal binding site and coding sequence to function. We only have experience using this part in B. subtilis, but it should function in most gram-positive organisms. It has not been tested and will most likely not work in E. coli. The promoter comes from the cis-regulatory region of the uracil phosphatase in Bacillus cereus. We functionally annotated the promoter ourselves using the DBTBS database motif search feature. (dbtbs.hgc.jp)
This part generates high expression in gram-positive organisms, and it may be possible to generate deleterious effects on the cell if used to express certain protein products.
We PCRed this part out of the pAD43-25 gram positive vector, acquired from the Bacillus Genetic Stock Center (bgsc.org), catalog number ECE166.
optimized (TA) repeat constitutive promoter with 13 bp between -10 and -35 elements
This part contains the pLac promoter and a ribosome binding site (BBa_R0010 + BBa_B0030). The Lac promoter is constitutively activated, but it can be repressed by the Lac I repressor from E.coli. This promoter is really useful to control a gene expression by inactivating it with LacI repressor. When LacI is absent the gene should be expressed, and in the presence of LacI, pLac is inhibited. So this part is really suitable for gene expression control by inactivation of pLac with LacI. We can use this part to control a failed feedback mechanism which induces the Lambda phage lytic cycle. In E.Coli, pLac is a part of the lactose operon. LacI is constantly expressed and represses the pLac. In the presence of Lactose, LacI is inhibited and pLac is active and allows the transcription of beta-galactosidase. Lactose can be substituted by IPTG (Isopropyl β-D-1-thiogalactopyranoside). This part already contains a strong RBS to begin directly a gene transcription.
Part:BBa_R0010 Part:BBa_B0030, Designed by Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003. Group: NCBS, Prather Lab.
It is the same as BBa_I0050. But the part seems not working so we make a new one, which the template is from iHKU team.
This is identical to BBa_I12007 exept that some base has been modifed to allow for additional ZFP repressors to bind.
Hybrid promoter constisting of two LacI O2 sites at the ends of the promoter and two recognition sites (OR2 and OR1) for the P22 c2 repressor. The promoter should function as a NOR gate.
a small fragment of OR3 remains.
Vector expressing eGFP from the CMV promoter with the BGH polyA tail and with a hygromycin resistance cassette
Compatible with the Plug'n'Play with DNA assembly system.
June Rhee, Connie Tao, Ty Thomson, Louis Waldman
Sequence for pTet inverting regulator driven by the TetR protein.
BBa_R0040 TetR-Regulated Promoter is based on a cI promoter. It has been modified to include two TetR binding sites and the BioBrick standard assembly head and tail restriction sites.
Lutz, R., Bujard, H., Nucleic Acids Research (1997) 25, 1203-1210.
This is the first part of the split infrared fluorescent protein (IFP1.4). To be used with IFP[2].
When fused to another coding sequence, insert a flexible linker
Promoter Family Member (U097O11+D050O22)
This is an IPTG-inducible promoter for use with gram-positive organisms. It requires a downstream gram-positive ribosomal binding site and coding sequence to function. We only have experience using this part in B. subtilis, but it should function in most gram-positive organisms. It has not been tested and will most likely not work in E. coli.
We found the functional annotation in the associated paper: Kaltwasser M., T. Wiegert, and W. Schumann. 2002. Construction and Application of Epitope- and Green Fluorescent Protein-Tagging Integration Vectors for Bacillus subtilis. Appl. Environ. Microbiol 68:2624-2628 The primers we used to extract it (sans biobrick ends) are as follow: Forward: AGAACAACCTCTGCTAAAATTC Reverse: AAGCTTAATTGTTATCCGCTC
We PCRed this part out of the pMUTIN-YFP gram positive vector, acquired from the Bacillus Genetic Stock Center (bgsc.org), catalog number ECE151.
Promoter Family Member (U085O11+D038O22)
This part is from the bacteriophage M13 genome, bp 1500-1547 in M13K07. It directs transcription of M13 gene III (BBa_M13503, BBa_M13003). Its identity as a promoter was described in Gene (1980) 11:129-148 based on -10,-35 homology and fragment of genome able to bind RNAP.
POPS regulated and Lac inverter system with strong RBS
The biobricks BBa_Q04121 and BBa_B0034 where used to build this biobrick
This is a derivate of BBa_K105027. Due to a point mutation in the TATA box region the efficiency of this minimal promoter is only 28 % of the natural yeast CYC1 promoter.
Promoter for recA gene - SOS and Ultrasound Sensitive
According to Vollmer, et. al in their 1998 Applied and Enviormental Microbiology paper, "Bacterial Stress Responses to 1-Megahertz Pulsed Ultrasound in the Presence of Microbubble", the promoter for the recA gene, a known SOS response gene, is upregulated 4 fold when cells are exposed to high frequency ultrasound and microbubble cavitation.
We did not see the desired response from ultrasound exposure that we expected from this promoter. However, we did not induce the same acoustic cavitation that Vollmer et. al. used to elicit a response from this promoter.
For selection of E. coli transformants, contains promoter, ampR gene, and terminator
Our microarray experiment showed that this gene was responsive to sound and ultrasound. Therefore, we cloned the promoter region located approximately 250 base pairs before the start codon for the cadB gene.
There was a relatively high basal level of expression of this gene that made this part difficult to incorporate into our lysis device design.
cloned from E. coli MG1655 genome located upstream of CadA and CadB genes
Generator of (CHE)Cholesterol esterase. Attached to placI, the part is inducible by IPTG and Lactose. It works better in Lactose.
This promoter normally represses the ColE2 activity gene, ceaB, in the ColE2 operon. To activate this promoter would require a RecA positive strain, as the protein LexA represses the promoter. To induce this promoter, an intercalating agent, mitomycin C, was utilized to induce the SOS response, and thus the expression of the genes under control of this promoter.
Lab strains of E. coli, such as Top10 from Invitrogen, are deficient in RecA so we required a RecA positive strain to induce this promoter. We used BL21 from Novagen.
This part comes from the ColE2 operon, which came from a plasmid.
This is a composite part containing the minimal promoter mCYC preceded by multiple consecutive binding sites for the LexA binding domain.
Composite part was taken via PCR from a synthetic plasmid containing the sequence. The plasmid came from Lawrence Berkeley National Laboratory.
Predicted by Lon Chubiz to be RBS sequence with strongest affinity for T7 ribosomes.
This part is the natural RBS from the sipC protein in Salmonella Type III Secretion.
This part was not designed with biobrick standard assembly in mind. This RBS has not been explicitly measured for expression rate using fluorescence or B-Gal assay.
A part used to quantify the AraC-RBS construct K259007 created by the BCCS-Bristol iGEM'09 team.
This part was designed with the specified biobricks from the parts registry. AraC occurs naturally in E.coli. GFP occurs in jellyfish species, RBS designed by the Anderson family and Terminator by Resma Shetty.
SPI-1 TTSS secretion-linked promoter from Salmonella
This promoter is activated when functional SPI-1 type III secretion systems are present and functional in Salmonella typhimurium 1344.
The promoter includes the binding site for the transcription factor InvF. This transcription factor is activated by the SicA chaparone when the secretion system is active.
This part was cloned from Salmonella typhimurium 1344 genomic DNA.
Simon St. Pierre extracted from S. Cervisiae genome.
PalcA, inducible Aspergillus nidulans promoter
A promoter that is inducible by alcohol, threonine can be used as an inducer.
Promoter Family Member (U085O11+D014O22)
Operator for RcnR(YohL) the negative regulator of niquel extrusion pump(RcnA) in E.coli
We needed the RcnR operator without its promoter, so it was necessary to predict the promoter and discard it from the sequence.
Mitochondrial targeting sequence for Aspergillus nidulans
Mitochondrial targeting sequence (MTS) for Aspergillus nidulans. A MTS is usually required for the entry of proteins and intermediary metabolites into mitochondria. The MTS is a DNA fragment encoding the N-terminal 60 amino acids of the AcuJ.
Compatible with the Plug'n'Play assembly standard. This MTS should be placed at the N-terminal of the gene of interest.
Aspergillus nidulans genomic DNA (AN6279)
The BBa_J23100 constitutive promoter with Lac systems inverter and strong RBS
The biobricks BBa_K266008 and BBa_B0034 where used to build this part
The AraC/pBAD promoter can be used with prokaryotic chassis to regulate expresion of proteins. When the AraC/pBAD promoter is placed upstream of your canditate translational unit (protein to be expressed) it allows control of the expression of the said protein. This is done via controlling the concentration of arabinose sugar in the environment/solution of bacterial growth. This composite part does not have an LVA degradation tag at its end, hence can be used with all prokaryotic assemblies.
The part was designed and assembled from the iGEM2009 kit with standard RFC10 assembly.
The AraC promoter is found naturally in E.Coli at 1.52 min/centisome in the clockwise orientation. More information can be found at [http://ecogene.org/geneinfo.php?eg_id=EG10054 EcoGene].
This is the GAL1 inducible promoter for use in Saccharomyces cerevisiae. It is repressed in the presence of glucose and induced in the presence of galactose. Raffinose is a neutral sugar source the neither induces nor represses the promoter. pGAL1 normally has an "all-or-nothing" response to galactose, but Christina Smolke has shown that knockout of the Gal2 gene gives pGAL1 a tunable, linear response to galactose.
From the Saccharomyces cerevisiae genome, via Caroline Ajo-Franklin.
Transcriptional terminator consisting of a 64 bp stem-loop.
Double Promoter (LuxR/HSL, positive / cI, negative)
This is a derivate of BBa_K105027. Due to a point mutation in the TATA box region the efficiency of this minimal promoter is only 43 % of the natural yeast CYC1 promoter.
LasR and 3OC12HSL regulated promoter with CI Operator 1 (binding site for lambda cI repressor)
This is the natural RBS for the sigE chaperone (BBa_J64025) in Salmonella Typhimurium LT2.
This part was not designed with compatibility to biobrick standard assembly in mind. This RBS has not been explicitly assayed for expression rate.
==What this part gives upon expression== This part encodes for FhuA directly as it is already in frame with a pBAD/Arabinose regulated promoter. Promoter Induction can occur with 0.1% Arabinose in the culture environment. FhuA is an E.coli Outer Membrane (OM) protein used for scavenging iron from the nutrient medium. For more information on FhuA visit the FhuA main page ( K259000 ). ==Potential uses of this Device== This part can be used also to create protein fusions by adding other proteins to it's 3' end. Protein fusion can be accomplished straightforwardly using the Bioscaffold-Linker biobricks K259003 . Protein fusions with FhuA may allow delivery of proteins to the OM of E.coli cells or inclusion into Outer Membrane Vesicles (see iGEM'09 Bristol-BCCS project).
The part was assembled using standard RFC10 assembly.
The arabinose promoter occurs naturally in E.coli. The RBS is designed from the Anderson family. FhuA also occurs naturally in E.coli.
RHLR/RHLI Inducible & LasR/LasI repressible Promoter
There is a 17bp spacer between the -35 and -10 regions and the binding site for RHLR/RHLI is 20bp long. I was able to fit the whole binding site in the spacer region because the last three base pairs of the -35 region and the first three base pairs of the RHLR/RHLI binding site are the same.
Double Promoter (LuxR/HSL, positive / P22 cII, negative)
Zif268-HIV binding sites + TEF constitutive yeast promoter
Promoter Family Member with Hybrid Operator (D001O21)
This promoter was isolated by taking 500bp upstream of the MF(ALPHA)2 gene of S. cerevisiae. This promoter will be activated when alpha factor is expressed in an alpha haploid yeast cell.
This gene is on the crick strand of the entered sequence.
This is the natural RBS associated with the sopA effector protein in the Salmonella SPI-1 system.
This part was not designed with compliance to biobrick standard assembly in mind. This RBS hasn't been explicitly tested in an expression assay such as GFP fluorescence or B-Gal expression.
PompR is a OmpR-controlled promoter which can be positively regulated by phosphorylated OmpR. This promoter is taken from the upstream region of ompC. Phosphorylated OmpR binds to the three operator sites and activates transcription. We insert PompR(BBa_R0082) sequences into the upstream region of our target gene gp35 through restriction-ligation method, which together is connected to vector PSB1C3 afterwards, and therefore we are able to regulate the expression of in through the control of light. Bxb1 gp35 is a serine integrase in Mycobacterium phage Bxb1. This intergrase can exclusively catalyze site-specific recombination. The dismiss of light singal can drive the expression of integrase, which alone can set a DNA sequence(there it means the promoter) flanked by oppositional attB and attP sites, thereby produce an inverted sequence flanked by attL and attR sites. As a consequence, the promotor moves on to express the toxic protein instead of the attractant. We insert the gp35 gene into the downstream region of the PompR promotor by means of 3A synthesis so that the expression of integrase can be regulated by light. In addition, we add a 6N promotor between PompR and gp35 to decrease basal integrase expression above flipping threshold level which could lower the efficiency of int translation. Meanwhile, we insert the recombined sites of attB and attP into the flanking sequences of our constitutive promotor (BBa_J23110 ) and so far we accomplish our reverse promotor system.
we add a 6N promotor between PompR and gp35 to decrease basal integrase expression above flipping threshold level which could lower the efficiency of int translation.
luxR operator (lux box) was deleted from R0065 and replaced with a BamHI hexamer.
added a bamHI site immediately upstream of -35 in place of lux operator
This RBS is part of the bacteriophage M13 genome, initiating translation of the gene VIII message (BBa_M13008). The part aligns with base pairs 1285-1300 in M13K07 genome. It was identified as the RBS for gene VIII message by Wezenbeek et al in Gene (1980) 11: 129-148
Gli1 binding sites + ADH1 constitutive yeast promoter
The Gli1 transcription factor binding sites on the ADH1 constitutive promoter. Transcription regulation should be constitutive plus regulated by YY1-binding transcription factors.
Promoter Family Member (U097O11+D014O22)
This is the second part of the split infrared fluorescent protein (IFP1.4). To be used with IFP[1].
K091146 is a modified version of the pLas' promoter (BBa_K091117) containing the addition of a luxR binding site between the -35 and -10 regions. This promoter is expected to be activated in the presence of PAI-1 and LasR and repressed in the presence of AI-1 and LuxR.
The Lux box was placed adjacent to the LasR binding site with the aim of minimizing cross-talk in the presence of both PAI-1 and AI-1; since the LasR site should have a higher affinity, we hope that the binding of PAI-1 will competitively exclude the binding of AI-1.
The LuxR binding site is from BBa_R0062 and the pLas' promoter is from the region upstream of the LasI gene in Pseudomonas aeruginosa.
Strong Expression level Constitutive promoter in yeast
Endogenous promoter for Alcohol Dehydrogenase
OmpR-P strong binding, regulatory region for Team Challenge03-2007
Contains the F1 consensus site from the OmpF regulator. Contains a LacI repressor consensus binding site between the -35 and -10 portions of a T7 core promoter region, and a TetR consensus binding site upstream of the -35 site.
The weaker binding repressor binds upstream of the core promoter (upstream of -35), while the stronger binds inbetween the -35 and -10.
Information for creation of this construct came from the following sorces: Yoshida et al. JBC 2006 Frank et al. JMB 1997 Rong et al. PNAS 1998 Merabet et al. Biochemistry 1995 Su et al. JBC 1998 Kedracka-Krok et al. JPC 1999 Kleinschmidt et al. Biochemistry 1988 Butler et al. JBC 1982 Jorgensen et al. JBC 1991 Hillen et al. JMB 1984 Head et al. JMB 1998
This is the natural RBS that drives the sipA effector protein in the Salmonella SPI-1 system
This part was not designed with compatibility to biobrick standard assembly in mind. This RBS has not been explicitly tested in an expression assasy such as B-Gal or GFP expression.
Promoter Family Member (U073O26+D062NUL)
Binds Maltose and stimulates transcription
Double Promoter (lacI, negative / P22 cII, negative)
This is a derivate of BBa_K105027. Due to a point mutation in the TATA box region the efficiency of this minimal promoter is only 16 % of the natural yeast CYC1 promoter.
Restriction sites of Sal I, Bam HI, Bgl II, and Mlu I in front of strong RBS
We designed new prefix(Sal I and Bam HI) and suffix(Bgl II and Mul I) for insertions of promoters and regulator binding sites in front of RBS.
This is the native ribosome binding site of ftsK, a DNA-translocating motor protein involved in cell division in Escherichia coli.
Virtual part to be added to coding sequence BBa_I742137.
low expression, constitutive promoter in yeast
Lac system inverter controlling the expression of LasR
The biobricks BBa_Q04121, BBa_B0034 and BBa_K266002 where used to build this biobrick
This part was designed for the Plug'n'Play with DNA assembly standard (RFC 80), therefore this part contains tails at the 5' and 3' ends as shown on the figure below. Please notice that mRFP1-NLS is not the same as BBa_K678030, BBa_K678031, or BBa_K678032.
From p71-mRFP1 of the Center for Microbial Biotechnology plasmid collection at the Technical University of Denmark. The NLS sequence was synthesized.
As per BBa_K180002, omitting the RBS and STOP biobrick parts (B0034 and B0015) for speed and ease of construction.
Assess if the RBS (B0034) sequence is needed prior to GFP (E0040) whilst still able to test function of part K180000.
An acid inducible promoter of the acid shock response (asr) gene, extracted from E. coli K12. This part can be used to generate pH based response in any biological system.
The sequence provided in literature indicated a consensus sequence for the Pho Box just before the transcription start site
This part was extracted from the genome of E. coli K12
Promoter Family Member (U049O11+D050O22)
Promoter Family Member (U061O11+D002O22)
This is a mid-high level constitutive promoter from the yeast gene TEF2.
Promoter Family Member (U097NUL+D050O22)
AraC operator, truncated to include araO1, araI1, araI2, c-amp1, and c-amp2 sites. This operator should *activate* transcription in the presence of AraC; b/c the operator lacks the araO2 site, there should not be araC-mediated repression.
enhanced green fluorescent protein for use in mammalian cells
This is the promoter activated by the transcriptional regulator XylR (BBa_I723136) in response to detection of environmental xylene.
Modification of biobricks part BBa_B0015
GoL - Primary plasmid (part 1)/RPS - Paper primary plasmid (part 1) [LuxR generator]
Part of the iGEM2009 Team:Southampton Game of Life primary plasmid.
Constitutive promoter selected to allow for continuous expression of C0062.
Full-length stationary phase osmY promoter
Truncated Forward Primer: 5'- GTT TCT TCG AAT TCG CGG CCG CTT CTA GGCT TAT GTT TTC GCT GAT ATC - 3' Total Length: 49 bp Annealing Length: 21 bp GC Content: 38.1% Melting Temperature: 49.5 degrees C hairpin deltaG: -2.66 kcal/mol self dimer deltaG: -99.97 kcal/mol Full Forward Primer: 5'- GTT TCT TCG AAT TCG CGG CCG CTT CTA GCTG GCA CAG GAA CGT TAT C - 3' Total Length: 47 bp Annealing Length: 19 bp GC Content: 52.6% Melting Temperature: 53.4 degrees C hairpin deltaG: -3.99 kcal/mol self dimer deltaG: -98.3 kcal/mol Reverse Primer: 5'-GTT TCT TCC TGC AGC GGC CGC TAC TAG TAT TGT TAA ATA TAG ATC ACA ATT TTG- 3' Total Length: 54 bp Annealing Length: 25 bp GC Content: 20.0% Melting Temperature: 46.4 degrees C hairpin deltaG: -2.86 kcal/mol self dimer deltaG: -99.44 kcal/mol heterodimer deltaG with Forward Primer: -99.44 kcal/mol heterodimer deltaG with Conserved Forward Primer: -99.97 kcal/mol
Artificial terminator, estimated %T~=25% * 6bp stem, 4nt loop * Bidirectional, estimated reverse %T~<10%
Bidirectional, with the reverse estimated to be less effective than the forward. Has a polyA tail of 3 residues. Designed to be a terminator with low efficiency.
R.reniformis luciferase + ADH1 terminator + CaUra3
Binding region for LasR protein (positive regulation)
"Analysis of the Pseudomonas aeruginosa Elastase (lasB) Regulatory Region". Lynn Rust, Everett Pesci, and Barbara Iglewski
fhuA - Outer membrane transporter for ferrichrome-iron
fhuA is an outer membrane (OM) protein that is found in the gram negative bacterium, E.coli. It is involved in the active-transport (energy-dependent uptake) of ferrichrome-iron from the extracellular environment. However fhuA is only able to bind ferrichrome-iron (i.e. ferrichrome iron is a ligand of fhuA protein). For the uptake to be completed fhuA needs to form a complex with another protein, tonB that carries out the energy-dependent uptake by using ATP as substrate to undergo the relevant conformational changes. This biobrick ONLY encodes for fhuA. fhuA is a beta-barell protein (22 anti-parallel beta-strands) and is composed of 747 aminoacids. This receptor protein may be used as an attachment point by some colicins. References: Locher K.P., Rees B., Koebnik R., Mitschler A., Moulinier L., Rosenusch J.P. and Moras D. (1998),Transmembrane signalling across the Ligand-Gated FhuAReceptor: Crystal structures of Free and Ferrichrome-Bound States reveal allosteric changes. Cell 95,771-778. Uniprot access number: P06971 Ecogene: http://ecogene.org/geneInfo.php?eg_id=EG10302
The coding sequence (CDS) was already in compliance with assembly standard 10 (RFC10).
fhuA is part of the chromosomal genome of E.coli. In particular it can be found at 3.61min and follows the clockwise direction.
Promoter, Standard (HK022 cI regulated)
[Note: This is the same part as R0050 except that the -10 and -35 sites and spacing have been changed to comply with BBa_S0001]. The pR regulatory region is a 98 base-pair sequence with standard BioBrick prefix and suffix sections on its ends. It contains two operator sites at which HK022 cI protein can bind cooperatively. The -35 and -10 regions from pR are included along with some flanking sequences. The cutoff for the 5' end of the regulatory region was placed just after the pM -10 region which promotes transcription of a gene upstream (thus the pM -10 region is not included). The cutoff for the 3' end of the regulatory region was placed immediately prior to the RBS.
Derived from STHK022N . Modified to comply with BBa_S0001: TTGACA-17N-GATACT Literature claims that there is no cross-talk between HK022 cI regulatory region, Lambda cI, 434 cI and P22 cI, but this has not been verified experimentally by the synthetic biology group at MIT.
This part is from the bacteriophage M13 genome, bp 1155-1201 in M13K07. It directs transcription of M13 gene VIII (BBa_M13508, BBa_M13008). Its identity as a promoter was described in Gene (1980) 11:129-148 based on -10,-35 homology and fragment of genome able to bind RNAP.
Zewei yan, Yichong wang and Junjie zhang
BBa_K1072004 is the conding region of α-acetolactate synthetase(α-ALS).
Double terminator with two copies of <bb_part>BBa_B0010</bb_part>
hGH poly A, terminator for mammalian cells
From plasmid N-terminal pFLAG-CMV from Sigma-Aldrich
optimized (TA) repeat constitutive promoter with 19 bp between -10 and -35 elements
Annexin V is member of the annexin family of Ca dependent proteins. Its function is not yet known. It binds to phosphatidylserine in a Ca-dependen fashion.
In combination with RBS B0032 there is no detectable expression of annexin V.
This promoter is sensitive to HasI binding
This part is just for upload test. It is meaningless
Promoter with standard -10 and -35 sites. A symmetric Zif23 dimer target site is after the -10 site.
pTet + 37 oC RNA thermometer + mCherry (LVA)
This part contains a promoter regulated by TetR protein. The Transcription is regulated by a 37 oC riboswitch. The protein codified is a RFP with a LVA tag.
The prefix sites are EcoRI and XbaI and the sufix sites are SpeI and PstI.
Obtained synthetically. Promoter pTet. Based on a fusion of a RBS and a Riboswitch. RFP with a LVA tag (Further information coming soon).
LacI Consensus Binding Site in sigma 70 binding region
Promoter Family Member (U097NUL+D026O22)
A Double Reporter (LacZ-alpha and GFP) loaded on pSB3C5. For more information please refer to BBa_I732403.
Yeast optimized superfolder GFP C-terminus (215-238)
LacZ constitutive promoter, without including operators.
This promoter is repressed by tetR and lacI; it contains two binding sites for TetR and one LacI binding site. this part is an AND-gate since transcription occurs only in the presence of both ATC and IPTG.
Also designed by Pallavi Penumetcha and Robert Cool.
This part consists of the R0040 tetR promoter with the addition of the R0010 lacI binding site.
This part is the full renilla luciferase. The split protein has also been submitted as biobricks: BBa_K1486021.
promoter for FabA gene - Membrane Damage and Ultrasound Senstitive
According to Vollmer, et. al in their 1998 Applied and Enviormental Microbiology paper, "Bacterial Stress Responses to 1-Megahertz Pulsed Ultrasound in the Presence of Microbubble", the promoter for the fabA gene, a known membrane damage response gene, is upregulated seven fold when cells are exposed to high frequency ultrasound and microbubble cavitation.
constitutive promoter which outputs .4 TIPs point mutants of wild type rrnB promoter P1
Promoter Family Member (U085O11+D062NUL)
optimized (A) repeat constitutive promoter with 18 bp between -10 and -35 elements
This RBS is part of the bacteriophage M13 genome, initiating translation of the gene XI message (BBa_M13011). The part aligns with base pairs 3900-3915 in M13K07 genome. It was NOT identified as the RBS for gene XI message by Wezenbeek et al in Gene (1980) 11: 129-148
A lac repressible T7 promoter. This promoter is similar to those found in the pET vectors with T7lac promoters. I mutated the -9 base to an G, which should make it less than 3% of the strength of consensus. The base numbering is as used by Studier and coworkers (JMB 1991, 219, 45-59). See BBa_R0183 for more information.
This is the DNA sequence of a Y-type hammerhead ribozyme that targets and cleaves the PenI repressor (Part BBa_C0074). This ribozyme has target-binding flanking arms adjacent to the Y-type hammerhead catalytic core and synthetic extended arms located towards the 5' and 3' ends. The catalytic core allows for the sequence-specific cleavage of sites within the coding sequence of PenI. Hammerhead ribozymes are able to cleave at any NUH within an RNA sequence, where H represent any nucleotide except guanine (Mir 2001), although with GUC as the hammerhead consensus cleavage site (Lieber, 1995). The binding of the flanking arms adjacent to the hammerhead catalytic core favours the recognition and cleavage of only one target site. Additional secondary structure within the flanking arms was detected to a small degree by using IDT OligoAnalyzer's Hairpin tool (Owczarzy 2008) as is depicted below. [image] The synthetic extended arms are designed to bind to the complementary extended arms of another similar PenI-cleaving ribozyme (Part _____), but do not contribute to any additional secondary structure as confirmed via the Hairpin tool in IDT's OligoAnalyzer (Owczarzy 2008), as both arms of a particular ribozyme only contain a purine and pyrimidine pair that do not bind to each other (in this case, A and G). The two-nucleotide extended arms are also staggered in number to avoid slippage in binding and bind to its complement pair at significantly higher melting temperatures than the adjacent flanking arms. This feature allows for the sequestering of the two ribozymes when both are present and significantly decreasing any ribozyme activity. Regions of complementarity between the two target-binding regions of both ribozymes are also present to ensure that the functional catalytic cores are not able to cleave their mRNA targets, even when bound. The characteristics of this expressed pair of ribozymes were considered to create a functional RNA-based XOR logic gate. References Mir, A. A., Lockett, T. J. & Hendry, P. Identifying ribozyme-accessible?? sites using NUH triplet-targeting gapmers. Nucleic Acids Res 29, 1906???1914 (2001). Lieber, A. & Strauss, M. Selection of efficient cleavage sites in target RNAs by using a ribozyme expression library. Mol. Cell. Biol. 15, 540???551 (1995). Owczarzy, R. et al. IDT SciTools: a suite for analysis and design of nucleic acid oligomers. Nucleic Acids Res 36, W163???W169 (2008).
Although this particular part is likely to only cleave the PenI repressor mRNA due to its sequence specificity, the simple in silico design of this ribozyme allows for the cleavage of virtually any RNA sequence containing a cleave site (NUH). This particular sequence was obtained by first scanning our target PenI mRNA for the optimal cut site GUC. The 10 nucleotides surrounding this cut site were obtained and the complement of this area was generated for the ribozyme flanking arms. The length of this region was decided by ideal melting temperature (51.4 degrees Celsius). Additional synthetic extended arms were subsequently added to the 5' and 3' ends of the sequence for binding to the complement ribozyme in an XOR gate. This region of non-pairing purines and pyrimidines was staggered in base repeats to avoid slippage of binding and was defined in length by significantly higher melting temperatures (approximately 68 degrees Celsius). Finally, the catalytic core of the Y-type hammerhead ribozyme was introduced at the appropriate region. Throughout this entire process, secondary was continually checked and minimized using IDT OligoAnalyzer's Hairpin tool (Owczarzy 2008). The complete ribozyme sequences was also BLASTed again the E.coli genome, which was our chassis and yielded insignificant results. Other factors such as differential length or target binding regions due to varying melting temperature and GC content were also considered during the in silico design of this ribozyme and its pair in the XOR logic gate. References Owczarzy, R. et al. IDT SciTools: a suite for analysis and design of nucleic acid oligomers. Nucleic Acids Res 36, W163???W169 (2008).
The target-binding flanking arms of the ribozyme were obtained from the complement of the PenI Repressor sequence.
This a flexible linker combined by two former linkers, which can be easily connect to the end of protein with overlap pcr.
when we chose a linker for our fusion protein，we would have wanted to use -gggsgggs- linker. But we found that such a tandem repeating linker is hard to work well with overlap PCR because the OE region should be unique. Therefore, we improved the original part by change the first GGGS to a carefully selected natural linker from linker database to avoid repeating sequence. Besides, we used the selected natural linker to adjust its length to about 50A.
The HasR gene is originally found in the Serratia marcescens. We obtained the gene sequence from the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/nuccore/X81195.2) and synthesised the gene.
Placement of the operator site was determined to optimize steric interference between C2003 and RNA polymerase.
This promoter was rationally designed and synthesized.
OmpF promoter that is activated or repressesed by OmpR according to osmolarity.
Promoter OmpF is activated by phosphorylated OmpR at low osmolarity, and is repressesed by phosphorylated OmpR at high osmolarity.In Escherichia coli, osmoregulation is mediated in part by the actions of such a two-component system consisting of EnvZ and OmpR. These proteins act to control the relative levels of the outer membrane porin genes, ompF and ompC. At low osmo larity, OmpF predominates in the outer membrane, while at high osmolarity the OmpF porin is replaced by OmpC.OmpF has a larger pore and a faster ??ow rate than OmpC. Reference: K. Mattison, R. Oropeza, N. Byers, L. J. Kenney, J Mol Biol 315, 497 (Jan 25, 2002).
The osmolarity range of OmpF sensing still have to be confirmed
Promoter OmpF is activated by phosphorylated OmpR at low osmolarity, and is repressesed by phosphorylated OmpR at high osmolarity.In Escherichia coli, osmoregulation is mediated in part by the actions of such a two-component system consisting of EnvZ and OmpR. These proteins act to control the relative levels of the outer membrane porin genes, ompF and ompC. At low osmo larity, OmpF predominates in the outer membrane, while at high osmolarity the OmpF porin is replaced by OmpC.OmpF has a larger pore and a faster ??ow rate than OmpC.
promoter to xylF without CRP and several binding sites for xylR
removed RBS crp binding and xylR binding sites
This is the whole regulatory region from the URA3 gene coding for OMP decarboxylase in S. cerevisiae budding yeast.
NCBI Sequence database: http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=Nucleotide&dopt=GenBank&val=172061
This promoter was isolated by taking 500bp upstream of the MFA2 gene of S. cerevisiae. This promoter will be activated when A factor is expressed in an A haploid yeast cell.
For this part an artifical Terminator is used (generally added to another parts). The promoter is a TetR repressible promoter.It is sequenced for pTet inverting regulator. Promoter is constitutively ON and repressed by TetR. TetR repression is inhibited by the addition of tetracycline or its analog part
Bidirectional, with the reverse estimated to be more effective than the forward. Has a polyA tail of 9 residues. High expression of lysozyme only is toxic to bacteria. BBa_R0040 (TetR repressible promoter) is based on a cI promoter. It has been modified to include two TetR binding sites. Constructed by DNA synthesis.
Enterobacteria phage T4 http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=29366675&from=66503&to=66997&view=gbwithparts modified E. coli thr terminator, replaced all A-T pairs in stem with C-G pairs BiblioPlus Extension Error fetching PMID 9092630:
This is the native ribosome binding site for sam5 (4-coumarate-3-hydroxylase) of the bacterium Saccharothrix espanaensis.
This is a virtual part to be added to coding sequence I742141.
Promoter for transcribing keys to act on locks based on Isaacs, Collins, et. al: "Engineered riboregulators enable post-transcriptional control of gene expression" KP3 absorbs three nucleotides from mixed site ("TACTAGAG"), so that the key has a 5 nucleotide spacer region (i.e. "TAGAG") between the transcription start site and the first nucleotide of the key. We are still looking into what the optimal size for the spacer region is. See also KP2 (BBa_J01007), and Key1 (BBa_J01008), Key2 (BBa_J01009), Lock1 (BBa_J01010), and Lock2 (BBa_J01011).
This is a LuxR-30C6HSL-independent version of R0065. All DNA upstream of the -10 box is from the constitutive promoter J23119. All DNA downstream from the -10 box down is from R0065. This results in the replacement of the Lux box region for a constitutive promoter region and the maintenance of the two cI operators downstream of the transcription start site.
This cassette provides the entire tetracycline resistance operon and will express TetR.
Promoter, Standard (luxR and HSL regulated -- lux pR)
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr Drew Endy
[Note: This is the same part as R0062 except that the -10 and -35 sites and spacing have been changed to comply with BBa_S0001]. Promoter activated by LuxR in concert with HSL The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes.This expression is up-regulated by the action of the LuxR activator protein complexed with the autoinducer, 3-oxo-hexanoyl-HSL. Two molecules of LuxR protein form a complex with two molecules of the signalling compound homoserine lactone (HSL). This complex binds to a palindromic site on the promoter, increasing the rate of transcription.
This promoter is based on the Vibrio fischeri quorum sensing gene promoters. Two genes LuxI and LuxR and transcribed in opposite directions as shown below. The original sequence from which the parts <bb_part>BBa_R0062</bb_part> and <bb_part>BBa_R0063</bb_part> were derived is shown in the picture below. <img src="<bb_file part=BBa_R0062>Image1.gif</bb_file>" width="614" height="362"> Modified to comply with BBa_S0001: TTGACA-17N-GATACT
GFP Reporter Device, Heat-Shock or IPTG Induced
Construct assembled to allow the quantitative analysis of the heat shock/LacI promoter/repressor system, BBa_K338002 . Produces GFP in response to low-temperature heat shock, more-so in the presence of LacI. GFP production is retarded in LacIq cells (or in the presence of +5mM glucose) about 4-6x. See K338002 for more detailed data.
The type of GFP used in this construct happens to have a very long half life. If growing cultures for fluorescence measurements, grow at 30??C - any higher will activate the promoter, causing GFP to be produced.
This device is regulated by UV irradiation
Zif268-HIV bs + LexA bs + mCYC promoter
The Zif268-HIV and LexA transcription factor binding sites on the mCYC minimal promoter (in that order.) Transcription regulation comes from Zif268-HIV and LexA-binding transcription factors.
dapAp is the promter of dapA gene in E.coli. DapA enzyme is part of diaminopimelate (DAP)biosynthesis pathway. DAP is necessary for peptidoglycan and lysine biosynthesis. dapAp prmoter has been previously studied: Acord04: Acord J, Masters M (2004). "Expression from the Escherichia coli dapA promoter is regulated by intracellular levels of diaminopimelic acid." FEMS Microbiol Lett 235(1);131-7. PMID: 15158272 It has been shown that dapAp driven expression is inhibited at hight DAP. We have tried to confirm this by
Medium RBS based on Ron Weiss thesis. Strength considered relative to <bb_part>BBa_B0030</bb_part>, <bb_part>BBa_B0032</bb_part>, <bb_part>BBa_B0033</bb_part>.
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix ("RBS-1" in figure 4-14 of thesis). No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info for this part: Brian Cho
cI repressor from phage TP901 infecting L. lactis
This promoter is a modified version of the Mnt promoter that is also responsive to TetR. The promoter should be repressed by Mnt repressor. It should also be repressed by TetR, and in the absence of Mnt repressor, should be induced by aTc.
Mnt repressor binds as a tetramer to two half-operator sites. Introduction of TetR binding sites between the Mnt promoter -10 sequence and the start site for transcription should allow for repression by TetR. Accordingly, the hybrid promoter was designed by 1) Remove all bases of mnt promoter 3??? to -10: gagtcgtattaattt will be replaced by tccctatcagtgata, and 2) truncate the second half of ptet -10 and mutate -35 so that it function to bind tetR (replace ttgaca with actgta), but is not a promoter, and 3) place the tetR1 and tetR2 binding sites 3??? to truncated mnt -10 sequence.
This part comes from the Mnt promoter and the TetR promoter
Promoter (luxR & HSL regulated -- lux pL)
The lux cassette of V. fischeri contains a left and a right promoter. The left promoter gives weak constitutive expression of downstream genes.This expression is down-regulated by the action of the Lux repressor, LuxR. Two molecules of LuxR protein form a complex with two molecules the signalling compound homoserine lactone (HSL). This complex binds to a palindromic site on the promoter, increasing the rate of transcription from Pr, repressing transcription from Pl
This promoter is based on the Vibrio fischeri quorum sensing gene promoters. Two genes LuxI and LuxR and transcribed in opposite directions as shown below. The original sequence from which the parts <bb_part>BBa_R0062</bb_part> and <bb_part>BBa_R0063</bb_part> were derived is shown in the picture below.Includes most of Lux reulatory region, including the LuxR binding site which activates the right promoter. A putative LuxR autorepression binding site is also identified adjacent to the -10 site of the right promoter. This 2nd site has 55% identity with the first site. Putative inverted repeats (of size 18-27 bp) also exist between these two sites (not marked above), which may represent binding sites for other regulatory proteins. <img src="<bb_file>Image01.gif</bb_file>" width="614" height="362"> Unspecified.
Promoter region from Acidithiobacillus ferrooxidans rus operon
This is the PP promoter taken from the P2 phage genome. It is an inducible promoter that is activated by a class of activators, including P2 ogr (I746350), PSP3 pag (I746351) and phiR73 delta (I746352). These different activators should cause different levels of activity of the PP promoter.
Cup-1 is a regulatory promoter that senses heavy metals such as copper, arsenic, cadmium, antimony, mercury, chromium, and selenium.
Comes from genomic DNA of saccharomyces cerevisiae.
This part is part of a family of Bioscaffold - Linkers. This part will help users interested in protein-fusion. The usage of the part will allow in frame assembly of 2(+) proteins by: *Removing the stop codons from the upstream protein. *Removing the scar created by the Assembly Standard 10 (RFC10) construction of 2(+) protein coding sequences. *Maintaining the start codon of the downstream protein. *Adding a flexible (Gly-Ser-Gly) linker between the two protein coding sequences. *Acting as a translational buffer if ligation is incorrect. ===Assembly Instructions=== *Create an Assembly Standard 10 fusion as below ; **Upstream Protein Part - Bioscaffold Linker - Downstream Protein Part** *Use the BioScaffold Specific Enzymes (i)Restrict with BpuEI to remove upstream scar (created between Upstream Protein - Bioscaffold) and stop codons from upstream protein part. (ii)Ligate and you will get the intermediate product; **Upstream Protein (w/out Stop codons) - Tyr - BioScaffold Linker - Downstream Protein** *Restrict with CspCI to remove downstream scar. *Ligate to get the final product; **Upstream Protein - Tyr-Gly-Ser-Gly- Downstream Protein** ====N.B.==== CspCI cuts with a 1 b.p discrepancy depending on the sequence. However this should not affect the restriction/ligation process as this has been taken into account and incorporated into the design. Even with the discrepancy involved the restriction/ligation process will still give the same product and in-frame fusion. The other restriction variants of CspCI have also been taken into account and relevant BioScaffolds are being produced to be tested. Updates will soon follow on this. This version accounts for cleaving DNA 12/10bp (top/bottom) on the 5' of the recognition sequence and 10/12bp (top/bottom) on the 3' site.
CspCI enzyme has different variations in the distance of restricting DNA from the recognition site.This biobrick is almost identical to part BBa_K259004 but modified to account for this discrepancy.
This part is completely designed de novo. This sequence does not occur naturally.
This RBS is part of the bacteriophage M13 genome, initiating translation of the gene IV message (BBa_M13004). The part aligns with base pairs 4204-4219 in M13K07 genome. It was identified as the RBS for gene IV message by Wezenbeek et al in Gene (1980) 11: 129-148
The Pseydomonas quinolone signal (PQS) as well as its precursor 2-heptyl-4-quinolone (HHQ)are very important in mechanisms controlling virulence in Pseudomonas aeruginosa. P. aeruginosa has a key role in Cystic Fibrosis (CF). PQS can initiate signalling for various processes like virulence, motility, and biofilm formation. A good number of these processes are facilitated by PqsR, which is a transcriptional factor. An activated PqsR will go to the nucleus of the cells and activate the respective genes. http://www.ncbi.nlm.nih.gov/pubmed/21539583 http://www.ncbi.nlm.nih.gov/pubmed/?term=pqsr-dependent+and+pqsr-independent+regulation+of+motility+and+biofilm+formation+by+pqs http://www.ncbi.nlm.nih.gov/pubmed/12393198
The sequence did not have any restriction sites to remove so it was immediately PCRed out of the genome and had the registry prefix and suffix added to it. It was then ligated into the pSB1C3 vector.
The source of this part is the genome of Pseudomonas aeruginosa PAO1 provided by the Robert Ryan lab in the Molecular Microbiology department of the University of Dundee, UK.
This is a 40-50A flexible separation for fusion domains with a SV40 NLS, and its codon is optimized for mammalian. It can be used to link different protein domains for fusion proteins locating in cell nucleus.
The codon is optimized for mammalian, and we insert a NLS to promote locating in cell nucleus. Besides ,we lengthen this linker to provide better separation of two domains. At last we avoid tandem repeat sequence(like GGGSPKKKRKVGGGS), which will facilitate fusing this linker to your domains via overlap PCR.
This part is improved from BBa_K1486003.
Modified lamdba Prm promoter (repressed by 434 cI)
Lamdba Prm promoter modified to be activated by lamda repressor (cI) and repressed by 434 repressor (cI)
The O-R1 region of 434 contained 14 base pairs as opposed to the 17 base pairs of the O-R3 site of lambda. Also, it was noticed that the O-R3 site of the lambda included part of the -10 site. Hence, to preserve the spacing and the -10 site, the three nucleotides that were in both the -10 site and the lambda O-R3 site were retained. The 14 nucleotides that were in the O-R3 site and not in the -10 site were replaced with the O-R1 site of the 434.
RBS (Elowitz 1999) -- defines RBS efficiency
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix. No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info for this part: Brian Chow
Promoter for cell division genes ftsI and ftsW
BBa_S03331is based on the reguatory region preceding cell division genes in the mra cluster and includes the biobrick head and tail restriction sites.
A promoter for the first nine genes of the Escherichia coli mra cluster of cell division and cell envelope biosynthesis genes, including ftsI and ftsW. Hara H, Yasuda S, Horiuchi K and Park JT. Journal of Bacteriology (1997) 179: 5802-5811
Promoter Family Member (U037O11+D062O22)
hygromycin cassette for Aspergillus nidulans
This E. coli promoter is positively regulated by arabinose. araC is the regulator protein, binding to arabinose prevents the repression of pBAD.
This part is similar to BBa_I0500, but it differs from that one in several point mutations in the sequence.
The 434 cI regulatory region sequence is a 89 base-pair sequence with the standard BioBrick prefix and suffix sections on its ends. 434 cI repressor protein, <bb_part>BBa_C0052<bb_part>, binds to it. This segment contains O-R1, O-R2, P-R, and P-RM -35.
same part as BBa_P1002 except in reverse orientation.
This is identical to BBa_R0051 exept that some base has been modifed to alow for additional ZFP repressors to bind
NOT Gate Promoter Family Member (D001O1wt1)
Biobricked version of Isaacs' riboregulator cis repressed lock, crR12.
Test RBS from T7 that may be used in E.coli
5' upstream sequence of qrr4 containing -12 and -24 regions (specific for Sigma factor 54)
>gi|156527546:c1833759-1833376 Vibrio harveyi ATCC BAA-1116 chromosome II, complete sequence
This is a single bp change from B0034. It's strength is approximately 35% that of B0034.
This actuator's purpose is to code for the output of the enzyme melamine deaminase. This enzyme eliminates an amine group from melamine to produce ammonia and ammeline and is able to remove another amine group at a rate 10 times slower than the first reaction to bring the compound to ammelide. The enzyme does not react to a measurable extent with this last compound, so it metabolizes melamine by consecutive and predictable deaminations. Its design comprises three components, which include a bicistronic ribosome binding site, the MDA gene of interest which codes from melamine deaminase, and a transcription terminator. The part needs a PoPS signal as an input in order to produce the desired enzyme and was designed to work in E. coli.
The TriA gene???s codons were modified from the original sequence and optimized for E. coli to a Codon Adaptation Index (CAI) of .76 (Puigb??, P., Guzm??n, E., Romeu, A. & Garcia-Vallv??, S. OPTIMIZER: A web server for optimizing the codon usage of DNA sequences. Nucleic Acids Research. 2007;35:W126???31.). After that, some palindromic and mRNA destabilizing sequences were removed to increase the stability of our DNA during synthesis. The sequence shown maintains a CAI of .76 and the same amino acid sequence as the original TriA gene.
The original gene coding for the protein of interest was isolated from a species of Pseudomonas (http://www.ncbi.nlm.nih.gov/nuccore/11890744). The sequences for the RBS and Terminator were sourced from BIOFAB (http://biofab.org/)
RBS specific to dedicated ribosomes. This RBS is a modified version of B0036. The spacing between the SD sequence and the start codon has been increased by 5 bases to give the same spacing as used by Brink et al. The 5 bases are the same as those used by Brink. That means they form the first five bases of an XbaI site and no negative affects of using these bases are known at present.
This is a TetR protein generator regulated by the constitutive promoter J23111. The protein has a LVA tag for degradation.
This a GFP reporter regulated by an allosteric intron responsive to theophylline.
The ligand response is alter from c-di-GMP to theophylline by merging a theophylline hammerhead ribozyme to the intron.
Novel gentamicin resistance gene. Derived from Genbank DQ208936, pTEX5500ts, kind gift of Barbara E. Murray. This, in turn was derived from Genbank AF016483. See pmid=16391062, pmid=9593155
XbaI site at 660 removed by mutating c->t which also improves codon usage. gatgatttttacagggatattttagagaacgaaatctac,P1014-XF gtagatttcgttctctaaaatatccctgtaaaaatcatc,P1014-XR
The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes.This expression is up-regulated by the action of the Lux repressor, LuxR. Two molecules of LuxR protein form a complex with two molecules the signalling compound homoserine lactone (HSL). This complex binds to a palindromic site on the promoter, increasing the rate of transcription.In thisversion, a binding site for cI dimer has been added: binding to this site dominantly represses transcription.
V. fishceri and phage lambda (courtesy of Ron Weiss)
Template for Building Primer Family Member
Using this promoter allows for the constant expression of a reporter gene or a selectable antibiotic.
This part was obtained from members of the Weiss Lab.
The ribosomal binding site for gene creC in the E Coli cre operon.
Equivalent part to r0040, but in BBb format. In house, part is referred to as pBca9145-Bca1091 {Ptet}. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR ca1091F/R on pSB1A2-r0040 (~100 bp, EcoRI/BamHI) Sub into pBca9145-Bca1089 (EcoRI/BamHI) Product is pBca9145-Bca1091 ---- ca1091F Forward EcoRI/BglII for Ptet part ttctggaattcatgAGATCTtccctatcagtgatagag ca1091R Reverse BamHI for Ptet part GtttGGATCCgtgctcagtatctctatc
Strong RBS based on Ron Weiss thesis. Strength is considered relative to BBa_B0031, BBa_B0032, BBa_B0033 and BBa_B0034.
Gli1 binding sites + TEF constitutive yeast promoter
The Gli1 transcription factor binding sites on the TEF2 constitutive promoter. Transcription regulation should be constitutive plus regulated by Gli1-binding transcription factors.
CMV promoter with inserted lac operator sites. Lac operator from POPI3Cat
This is the native ribosome binding site of crtE (geranylgeranyl pyrophosphate synthase) from Pantoea ananatis (formerly Erwinia uredovora) DSMZ 30080 (ATCC 19321) Accession: D90087.
This is a virtual part to be added, blunt ended, to the coding sequence.
Pantoea ananatis (formerly Erwinia uredovora) DSMZ 30080 (ATCC 19321) Accession: D90087.
NOT Gate Promoter Family Member (D001O22)
R.reniformis luciferase + ADH1 terminator + Kanamycin resistance
Rock-paper-scissors - Rock secondary plasmid [rhl pR, LacI generator]
Regulated expression of LacI (C0012) by Rhl pR (R0071).
See relevant basic biobrick parts (R0071, B0034, C0012, B0015)
Sequence for sigma 32 transcription factor
This part is a correction to [http://parts.igem.org/Part:BBa_M36888 BBa_M36888] which has an illegal PstI site which makes it incompatible with BioBricks / RFC10. iGEM Teams can use this alternative sequence which contains none of the BioBrick restriction enzyme sites. Amino acid No. 202, Glysine has been mutated from CAG to CAA to remove the PstI site. The remainder of the sequence remains unchanged.
This part was checked for BioBrick restriction enzyme sites which would otherwise make it unsuitable for use by iGEM teams.
This part is a correction to [http://parts.igem.org/Part:BBa_M36888 BBa_M36888].
this is a modified version of the LacIQ promoter that should bind LacIQ tighter than the original.
This is a modified version of the LacIQ promoter.
This is suggested to be an adhesion responsive promoter. It contains the binding site for CpxR-P (phosphorylated CpxR). CpxR is one member of the two-component Cpx response to envelope stress. Briefly, envelope stress causes CpxA, an inner-membrane histidine Kinase to Phosphorylate CpxR, allowing it to bind to CpxR-P responsive regions and regulate downstream target genes. See 'Surface sensing and adhesion of Escherichia coli controlled by the Cpx-signaling pathway', Karen Otto and Thomas J. Silhavy, PNAS, Vol. 99., No, 4., 2287-2292. 2002.
This part comes from the genome of E.coli TR235, as used in the PNAS paper of Otto and Silhavy mentioned previously. TR235 is K-12 MC4100 containing a pCpxR-LacZ fusion inserted at the (Lambda)att site, plus some other modifications (see Otto and Silhavy). The part is a portion of the CpxR responsive region used in these LacZ fusions.
Promoter Family Member (U049O11+D062NUL)
A Double Reporter (LacZ-alpha and GFP-AAV) loaded on pSB3K5. For more information please refer to BBa_I732402.
pyrG-DR cassette, for selection of Aspergillus nidulans
pyrG-DR cassette with directed repeats, for selection of Aspergillus nidulans
Promoter Family Member (U073O26+D062O61)
constitutive promoter with (C)10 between -10 and -35 elements
With 10 C repeats between the -10 and -35 elements, the distance between the two elements is optimal for the sigma factor to bind to the promoter. Thus, the promoter should be in the 'on' state.
PCR mv028/mv029 on rrnB native sequence (721bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112118 ----------------------------------------------- mv028 Forward Biobricking of rrnB P1 promoter cgataGAATTCatgAGATCTatgcactaaaacgtatcctacgcccg mv029 Reverse Biobricking of rrnB P1 promoter cgttaGGATCCcgccttcccgctacagagtcaagc
This is the weak version of the consensus RBS for Bacillus subtilis. The sequence is present in front of some B. subtilis operons, but is expected to give weaker expression than the consensus sequence. The consensus sequence AAAGGAGG (BBa_K090505) was changed to AGAGGTGG for this part.
The part must be 8 nucleotides away from the first methionine of the coding sequence, and so we added 'TGT' downstream of the actual binding sequence: AGAGGTGG. This, combined with the truncated non-CDS BioBrick prefix scar, gives exactly 8 'spacer bases' between the 3' end of the RBS and the start of the coding sequence.
Promoter Family Member (U085O11+D062O22)
gentamycin resistance cassette (aacC1 gene)
The aacC1 gene conveys resistance to gentamycin only. It is derived from a Tn21 series transposon (Wohlleben89) found on the R1033 plasmid (Pseudomonas aeruginosa). This part is created by PCR from the pJAT8 plasmid (Keastling lab) which in turn is derived from pJN105 (Newman and Fuqua).
pJAT8 plasmid edited with primers: GTT TCT TCG AAT TCG CGG CCG CTT CTA GAG ccttgtcgccttgcgtataa,aacC1-F GTT TCT TCC TGC AGC GGC CGC TAC TAG TA atctcggcttgaacgaattg,aacC1-R
This is the promoter for molecular chaperone htpG. It is active during the heat shock response.
This is a correction to part [http://parts.igem.org/Part:BBa_J45504 BBa_J45504], also the htpG Heat Shock Promoter. The original part contains a fragment of the coding sequence for molecular chaperone htpG (base pairs >287) which can be confirmed by [http://blast.ncbi.nlm.nih.gov/ BLASTing the sequence]. This part contains only the first 287 bases of the original part which consist solely of the htpG promoter. You can see the difference between the two sequences in the image below.
An 328 bp promoter region, which can be assembled with urea binding proteins to activate the downstream gene expression.
pUreD is a urea responsive element that should be turned on by the complex of UreR and urea.
This is a component of urea sensor cloned from Proteus mirabilis.
Promoter Family Member (U073O11+D050O22)
This is the natural RBS from the sopD gene in Salmonella Type III Secretion.
Promoter Family Member with Hybrid Operator (D001O47)
This is a TetR protein generator regulated by the constitutive promoter J23106. The protein has a LVA tag for degradation.
This is the PO promoter taken from the P2 phage genome. It is an inducible promoter that is activated by a class of activators, including P2 ogr (I746350), PSP3 pag (I746351) and phiR73 delta (I746352). These different activators should cause different levels of activity of the PO promoter.
The source of the DNA is the P2 phage genome.
This promoter can be repressed by lacI protein and induced by either IPTG or arabinose.
IPTG has better effect on this promoter than arabinose
Promoter Family Member with Hybrid Operator (D001O16)
This is the naturally-occurring RBS found in the regulatory region of the DntA gene.
'''''This does not physically exist as a basic part due to time constraints; it was cloned as part of composite part which incorporates all of the components necessary for expression of a reporter gene under the control of DntR'''''. For this reason, the ends of the supplied sequence may not accurately represent the boundaries of the functional unit.
Cloned from a historical plasmid based on pQF52 carrying genomic sequences from ''Burkholderia cepacia''.
Promoter, Standard (434 cI regulated)
[Note: This is the same part as R0052 except that the -10 and -35 sites and spacing have been changed to comply with BBa_S0001]. The 434 cI regulatory region sequence is a 89 base-pair sequence with the standard BioBrick prefix and suffix sections on its ends. 434 cI repressor protein, <bb_part>BBa_C0052<bb_part>, binds to it. This segment contains O-R1, O-R2, P-R, and P-RM -35.
Wild-type promoter modified to comply with BBa_S0001: TTGACA-17N-GATACT
Positively regulated, OmpR-controlled promoter. This promoter is taken from the upstream region of ompC. Phosphorylated OmpR binds to the three operator sites and activates transcription.
The promoter includes the three OmpR binding sites: C1, C2, and C3. The promoter starts at -107 and goes up to the transcriptional start site at +1. An alternate version, BBa_R0083, cuts out the C2 and C3 sites. The efficacy of this promoter versus the truncated ompC upstream region (BBa_R0083) or the ompF upstream region (BBa_R0084) is currently unknown.
This is the -20 to -1 region of T7 gp0.5, which contains the native RBS region for this gene. The physical instantiation of this part does not exist. This part is not compatible with Standard Biobricks assembly methods, as the scar created during assembly would interrupt the spacing between the RBS and the start codon of the CDS. Individuals wishing to use this RBS sequence in their assembles should refer to part B2103.
The -20 to -1 region was chosen to be representative of the RBS region of the T7 genes for the following reasons: 1) The Shine-Delgarno region is contained within this defined region for all T7 genes. 2) Current literature suggests that when bound to the Translation Start Site (0), the Ribosome covers positions -20 to +13 of the gene.
Genome of wild-type bacteriophage T7, positions 1476-1495. Includes the -20 to -1 region of gp0.5.
A lac repressible T7 promoter. This promoter is similar to those found in the pET vectors with T7lac promoters. I mutated the -10 base to a G, which should make it about 4% of the strength of consensus. The base numbering is as used by Studier and coworkers (JMB 1991, 219, 45-59). See BBa_R0183 for more information.
The -10 mutation from consensus was included to weaken the consensus promoter as described by Imburgio and co-workers (see BBa_R0183 for more information)
This sequence is based on the T7lac promoter described by Dubendorff and Studier (JMB 1991, 219, 45-59). The only modification is the mutation at -10.
E. Coli CreABCD phosphate sensing operon promoter
CreABCD phosphate sensing operon promoter taken from RegulonDB.
Lux pL controlled LuxR+Lux pR autoinduceing luxI(lva tag)+Lux pR controlled Ndh
Yichong wang, Zhiwei li, Xihao liao and Junjie zhang
BBa_K1072003 is a composite device, including LuxI，LuxR and Ndh genes. LuxR， controlled by Lux pI, generate LuxR protein, which will combined with AHL and activate Lux pR promotor. that complex increases the transcriptional rate of the lux pR promotor while also decrease the transceiptional rate of the Lux pL promoter. LuxI is the generator of AHL controlled by Lux pR promoter. Ndh was activated by AHL and LuxR complex and yields H2O2;
This promoter was isolated by taking 500bp upstream of the STE3 gene of S. cerevisiae. This promoter will be activated when alpha factor is expressed in an alpha haploid yeast cell.
This part was designed for the Plug'n'Play with DNA assembly standard (RFC 80), therefore this part contains tails at the 5' and 3' ends as shown on the figure below. Please notice that mRFP1-module is not the same as BBa_K678031 or BBa_K678032.
Promoter Family Member (U037O11+D026O22)
RBS.3 (medium) -- derivative of BBa_0030
Weak1 RBS based on Ron Weiss thesis. Strength is considered relative to <bb_part>BBa_B0030</bb_part>, <bb_part>BBa_B0031</bb_part>, <bb_part>BBa_B0033</bb_part>.
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix ("RBS-2" in figure 4-14 of thesis). Contact info for this part: Brian Chow
The T7 promoter should only produce PoPS when the T7 polymerase is also being expressed.
LasR and 3OC12HSL regulated promoter with a single Tet Operator (binding site for TetR repressor)
NOT Gate Promoter Family Member (D001O44)
Promoter Family Member with Hybrid Operator (D001O26)
Strong transcriptional terminator consisting of a 20 bp stem-loop that has been engineered to be bidirectional.
Suggested by Sri Kosuri and Drew Endy as a high efficiency terminator. The 5' end cutoff was placed immediately after the TAA stop codon and the 3' end cutoff was placed just prior to the RBS of Gene 1.4 (before AAGGAG). Subsequently mutations were introduced to make the terminator bi-directional (i.e., AAAAAA insertion on 5' side of the stem loop). Additional mutations were introduced on the 3' side of the stem loop to increase the number of T's and eliminate any promoter -10 site that might be present to avoid initiation of transcription of whatever is downstream. Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator.
Promoter Family Member (U061O11+D038O22)
Partial DLD Promoter from Kluyveromyces lactis
Promoter Family Member (U097O11+D002O22)
BGH poly A, terminator for use in mammalian cells
SV40 poly A, terminator for mammalian cells
SV40 poly A, terminator for use in mammalian cells
Promoter, Standard (p22 cII regulated)
[Note: This is the same part as R0053 except that the -10 and -35 sites and spacing have been changed to comply with BBa_S0001]. The p22 cII regulatory region sequence is a 97 base-pair sequence with the standard BioBrick prefix and suffix sections on its ends. p22 cII repressor protein, BBa_C0053, binds to it. This segment contains O-R1, O-R2, a fragment of O-R3, the -35 of P-RM, and P-R (-10 and -35 from Tom Knight)
Modified from wild-type to comply with BBa_S0001: TTGACA-17N-GATACT
This hybrid promoter uses the LsrA promoter that is capable of being repressed by LsrR - induction can occur with phospho-AI-2. Binding sites for cI repressor also occur. As a result the promoter is off in the absence of AI-2 and on in the presence of AI-2, but always off in the presence of cI.
The promoter region for LsrA should remain unaltered while cI binding sites (OR1 and OR2) are introduced downstream. The OR2 sites with cI bound should not allow transcription. The cI binding sites have a natural 6 bp spacer between them. It would be desirable to position the cI binding sites immediately downstream of the -10 region of the LsrA promoter. However, experimental evidence for the transciption start (+1) appears to be lacking.
The Missouri Western/Davidson team designed primers for LsrA and extracted the DNA from E. coli MG1655. The CI portion was pulled from the registry as part number R0051.
optimized (A) repeat constitutive promoter with 17 bp between -10 and -35 elements
This RBS is part of the bacteriophage M13 genome, initiating translation of the gene I message (BBa_M13001). The part aligns with base pairs 8252-8267 in M13K07 genome. It was identified as the RBS for gene II message by Wezenbeek et al in Gene (1980) 11: 129-148
Yeast optimized sfGFP N-terminus (1-214)
K338001+R0011: Heat Shock Promoter + LacI Regulated Promoter
Fused heat shock promoter with lacI regulated promoter. It possesses the characteristic of both heat shock promoter and LacI.
It was designed to address the issue of a leaky promoter in front of a bacterial gene in an effort to create a genetic switch just for growth/cloning
PTAC promoter followed by an RBS, green fluorescent protein and stop codon, for testing the effectiveness of the PTAC hybrid promoter designed by iGEM2009 Team:Southampton.
Inclusion of the RBS and stop sequence to ensure correct functionality observed.
This is the naturally-occurring RBS found in the regulatory region of the DntR gene. gataaaaacaagaggaaaacaa
pyrG cassette, for selection of Aspergillus nidulans
Double terminator + Constitutive promoter + Strong RBS
This part contains a double terminator and a constitutive prokaryotic promoter and a strong RBS (BBa_B0014 + BBa_J23100 + BBa_B0030). It is a simple promoter in the goals to begin gene transcription. For Sup???biotech-Paris (2009) team this part is used when adding to a recombinant lambda phage and to begin the transcription of synthetic genes inside the Lambda phage genome.
BBa_B0014 : Designed by Reshma Shetty Group: Registry (2003-07-16). BBa_J23100 : Designed by John Anderson Group: iGEM2006_Berkeley (2006-08-04.) BBa_B0030 : Designed by Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003. Group: NCBS, Prather Lab.
Hybrid promoter (trp & lac regulated -- tac pR)
The Ptac promoter is a functional hybrid promoter, derived from the trp and lac promoters, that are regulated by trp and lac [1]. The Ptac sequence has been extracted by PCR from the commercially available plasmid pMAL-pIII from New England Biolabs (N8101S), with the addition of the biobrick prefix and suffix sequences. [1] Proc. Natl. Acad. Sci. USA, Vol. 80, pp. 21-25
Sequence includes lacIq and Shine-Dalgarno sequence from pMAL-pIII prior to the Ptac promoter to ensure correct functionality of the promoter.
R0051 was modified to contain consensus -35 and -10 sequences. The CI operator OR1 was moved from it's original position (overlapping the -10) to between the -35 and -10. Fortuitously this results in the optimal (for high expression) 17bp spacing between the -35 and -10 sites.
Promoter Family Member (U073O11+D062NUL)
PgolTS-golS-PgolB-rbs-tetR-Ter-PtetO-rbs-rfp-Plac-rbs-tetR-Ter-Pcons2-rbs-lacI-Ter It is a test of our suicide circuit for gold ions.
PgolTS-golS-PgolB, tetR, PtetO, rfp, Plac, tetR, Pcons2, lacI, Ter
RBS.1 (strong) -- modified from R. Weiss
Strong RBS based on Ron Weiss thesis. Strength is considered relative to <bb_part>BBa_B0031</bb_part>, <bb_part>BBa_B0032</bb_part>, <bb_part>BBa_B0033</bb_part>.
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix ("orig" in figure 4-14 of Ron Weiss thesis). No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info Brian Chow
Shamik Mascharak, Justo Caballero and Sibel Sayiner
The rpoH gene codes for sigma 32 (alternatively sigma H, RpoH) transcription factor, a component of RNA polymerase sigma 32 holoenzyme. This 32-kD stress factor belongs to the sigma-70 factor family and is involved in intracellular temperature regulation, initiating bacterial transcription of various chaperone proteins and proteases in response to sudden increases in ambient temperature. The gene is currently classified as essential, due to the fact that rpoH deletion mutants fail to grow at standard incubation temperatures. This part may be employed in heat shock-to-PoPs sensors by coupling expression of the rpoH gene to action of sigma 32-inducible promoters such as the htpG heat-shock promoter (part#: BBa_J45405). Transcriptional activation by rpoH transcripts is possible both within and without the sensor genetic circuit, as sigma factors can diffuse around the nucleus.
Designs incorporating this part into a feedback amplification loop must beware of likely rpoH-deletion mutants. This is due to overproduction of sigma 32 under leaky promoters such as htpG (BBa_J45405). Less deletion mutants in E. coli are noted when plasmids containing rpoH are cloned at higher temperature. Consider two facts before using this part. First, rpoH transcript activity is reduced under excess heat shock protein production. As such, feedback loops using rpoH should employ weaker bicistronic ribosome binding sites so as to optimize heat-shock response. Second, sigma 32 is turned over rapidly, with a half-life under 1 minute.
The particular part described here is specific to the genome of W3110 strain E. coli.
This part is from the bacteriophage M13 genome, bp 4055-4103 in M13K07. It directs transcription of M13 gene IV (BBa_M13504, BBa_M13004). Its identity as a promoter was described in Gene (1980) 11:129-148 based on -10,-35 homology and fragment of genome able to bind RNAP.
This is a modified version of the LacIQ promoter tha should bind LacIQ tighter than the original.
Phns in BBb1 format. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
Construction of [<Phns>] Basic Part K112701 PCR dv001/dv006 on Phns from E. coli K12 MG1655 (~342 bp, gp = A) PCR dv005/dv003 on Phns from E. coli K12 MG1655 (225 bp, gp = B) PCR dv004/dv002 on Phns from E. coli K12 MG1655 (~149 bp, gp = C) --------------------------------------------------- PCR dv001/dv003 on A+B (355 bp, gp = D) --------------------------------------------------- PCR dv001/dv002 on C+D (697bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+697) Product is pBca1256-K112701 ----------------------------------------------- dv001 Forward Biobricking of [<Phns>] CgATAgaattcATGagatctGAAATATAGCTGTGCCATCAGCC dv002 Reverse Biobricking of [<Phns>] cagtcggatccGCACGAAGAGTACGGATG dv003 Removing the 2nd EcoRI site in [<Phns>] (R) GCCAGGAATGTAAGGgATTCAAAATTGTTC dv004 Removing the 2ND EcoRI site in [<Phns>] (F) GAACAATTTTGAATcCCTTACATTCCTGGC dv005 Removing the FIRST EcoRI site in [<Phns>] (F)CGCTTAATAGGGgATTCTCGTAAACAC dv006 Removing the FIRST EcoRI site in [<Phns>] (R)GTGTTTACGAGAATcCCCTATTAAGCG
HasI is the sigma factor under the has operon system
The HasI gene is originally found in the Serratia marcescens. We obtained the gene sequence from the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/nuccore/X81195.2) and synthesised the gene
consensus Kozak sequence and start codon
designed such that fusion using fusion bricks assembly method leads to in frame translation
consensus Kozak and start built from oligonucleotides
This RBS is part of the bacteriophage M13 genome, initiating translation of the gene VI message (BBa_M13006). The part aligns with base pairs 2840-2855 in M13K07 genome. It was identified as the RBS for gene VI message by Wezenbeek et al in Gene (1980) 11: 129-148
Transciptional regulator reliant on screams of someone, somewhere in trouble.
DNA sample - Taken from Clark Kent's Coffee Mug at the Daily Planet