Promoter Family Member (U037O11+D038O22)
Double Promoter (lacI, negative / P22 cII, negative)
Promoter Family Member (U085O11+D050O22)
Promoter Family Member (U097O11+D038O22)
Promoter Family Member with Hybrid Operator (D001O17)
modified Lutz-Bujard LacO promoter,with alternative sigma factor σ28
Lac and sigma 28 conserved binding sites
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.
Promoter Family Member with Hybrid Operator (D001O61)
Generator of (CHE)Cholesterol esterase. Attached to placI, the part is inducible by IPTG and Lactose. It works better in Lactose.
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.
Arabinose controlled protein degradation device
The purpose of this system is to control the expression of sspB, the degradation controller. The system is inducible by arabinose, therefore we can alter the arabinose levels to regulate the degradation of proteins with sspB degradation tag
sspB Biobrick is inserted after araR promoter+rbs system
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.
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.
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.
LacI Consensus Binding Site in sigma 70 binding region
Vikram Vijayan, Allen Hsu, Lawrence Fomundam
Constitutive Promoter, High Transcription
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.
we synthesize the OR promoter by replacing OR3 binding site of lambda CI with OR1 binding site of CI434.
making sure CI434 protein can repress CI's transcription.
similar to lambda lysis/lysogenic regulatory region.
In native e.coli, this promoter is located upstream of FecA cds. Regulated by FecI protein, which is in turn regulated by FecA.
Promoter Family Member (U073O11+D062O22)
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.
Promoter Family Member (U049O11+D002O22)
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.
Luminesensor repressible recA408 promoter
recA promoter in 408 form.recA promoter belongs to the SOS regulon family, and promotes in E.coli transcription of recA protein, which forms filament when bound to ssDNA and trigger the self-cleavage of LexA protein.recA408 have one SOS box, which binds to the dimerized LexA protein and cause inhibition of the transcription of downstream gene.
The original RecA was mutated to 408 form in order to be responsive to 408 mutant form of LexA DNA binding domain.
Fusion protein of SpyCatcher and RPD3 in an expression cassette
modified Lutz-Bujard LacO promoter,with alternative sigma factor σ32
Lac O and sigma 32 binding sites conserved sequences
Promoter Family Member (U049O11+D062NUL)
A Double Reporter (LacZ-alpha and GFP-AAV) loaded on pSB3K5. For more information please refer to BBa_I732402.
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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.
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].
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.
This promoter can be induced by IPTG. Wild type LacI binding site is replaced with a perfect palindromic "oid" operator sequence which increases the repression by LacI. This biobrick is an enhancement of Biobrick BBa_K090501 which has the sequence from pmutin2.
pmutin2's genbank accession number is AF072806. Although there is no sequence information for pmutin4, it can be derived from pmutin2's sequence information by changing the O1 lacI binding site with the "oid" sequence.
This sequence is taken from pmutin4 integration vector. pmutin2's lacI binding site was changed with the "oid" sequence to create pmutin4.
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.
Double Promoter (TetR, negative / P22 cII, negative)
improved for transcription factor BBa_J34807 binding
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 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
This part comes from the genome of ''E.coli'' K12 strain MG1655.
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.
Lac O and sigma 28 binding sites' conserved sequences
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.
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 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.
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
Lac O and sigma 32 binding sites' promoter sequences
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
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
Repressible E. coli promoter as annealed oligos
This is a part used as a building block for E. coli driven expression that is repressible by a synthetic protein (for more information visit 2015.igem.org/Team:Pitt). In our project, we used this promoter to detect proteases that could cleave the synthetic repressor, and so we named the promoter "pProt". This part uses a special cloning method with the following synthesized nucleotides: pProtsense: aattcgcggccgcttctagattgacaaacaagatatcttaaatgaaaatacaagaaatcttaaatata pProtantisense: ctagtatatttaagatttcttgtattttcatttaagatatcttgtttgtcaatctagaagcggccgcg The following method can be used to insert pProt before a desired Biobrick part (which should contain an RBS at minimum and preferably a cds and terminator): 1. Cut the desired plasmid with EcoRI and XbaI. Purify the plasmid with a method of your choice (both gel purification and PCR purification kits from Qiagen serve to remove the short undesired oligonucleotide from the digestion). 2. Be sure that your oligos have 5' phosphorylation (treat unphosphorylated oligos with T4 Polynucleotide Kinase). Then, anneal the two oligos by slowly cooling from 98C to room temperature. 3. Finally, ligate the cut plasmid with the annealed oligos by using a 1:10 molar ratio of plasmid to insert. 4. Transform the ligation reaction into E. coli, and plate. 5. Pick colonies, and verify the success of the cloning with sequencing or PCR. (Doing a diagnostic digest is not recommended as the size of the original plasmid and the desired plasmid are very similar.) 6. Add a composite part to the iGEM registry by using this part with the original protein coding part, checking the box to create without a scar, as this part contains a modified scar.
When ligating with a cut plasmid, this leaves a modified Biobrick scar between the pProt promoter and original Biobrick part.
Synthesized oligonucleotides as described on main page.
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.
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.
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.
Ptet-rbs-rfp-Ter-Plac-rbs-tetR-Ter. It produces rfp when there is enough lacI or less lactose.
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.
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.
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
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
37℃ induced RBS + tetR + double terminator
It is a transcriptional unit including 37℃ induced RBS + tetR + double terminator. It can be added various promoter to adapt your conditions.
To regulate following genes by temperature. To maintain cells at 37?? C ↑ : the tetR protein be generated and repress Ptet's circuit. To release cells at 37?? C ↓ : the tetR protein not be generated and do not repress Ptet's circuit.
the 37℃ induced RBS cames from iGEM08_TUDelft
modified Lutz-Bujard LacO promoter,with alternative sigma factor σ24
Lac O and sigma 24 binding conserved sequences
This promoter sequence (-80 to +1) is responsive to the transcription enhancer glnG.
Promoter Family Member (U037O11+D014O22)
This promoter is designed to bind to C2003.
The placement of the operator site was designed to be near optimal for steric interference between C2003 and RNA polymerase.
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.
Ptet-rbs-rfp-Ter. It produces rfp when there is less tetR.
---- 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
Promoter Family Member (U073O11+D062NUL)
Promoter Family Member with Hybrid Operator (D001O27)
This promoter can be repressed by lacI protein and induced by either IPTG or arabinose.
IPTG has better effect on this promoter than arabinose
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.
Promoter Family Member (U073O11+D014O22)
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.
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.
sense TWO INPUTS, activation by AHL and repression by LacI.
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.
This is a modified version of the LacIQ promoter tha should bind LacIQ tighter than the original.
This is a modified version of the LacIQ promoter
Promoter Family Member (U097NUL+D014O22)
A Double Reporter (LacZ-alpha and RFP) loaded on pSB3C5. For more information please refer to BBa_I732402.
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.
mutant promoter of lacI gene, which enables high level of lacI protein expression
point mutation made from E. Coli genomic lacI gene promoter
LacO and Sigma 24 binding conserved sequences
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.
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
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.
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
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 Family Member (U037O11+D062O22)
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
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.
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
MicF is a non-coding RNA which regulates expression of the ompF (outer membrane porin F) gene in E. coli. MicF binds to the 5??? untranslated region of ompF mRNA, to prevent translation and promote degradation. MicF can be used together with BBa_K176601 to regulate protein expression.
The transcribed MicF only consists of the last 93 nt of BBa_K1766000. The preceding region was added in consideration of the genomic sequence.
BBa_K1766000 is based on the micF gene found in Escherichia coli. The BioBrick??? was created from a gBlocks?? gene fragment synthesized by Integrated DNA Technologies.
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.
Promoter Family Member (U073O11+D026O22)
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].
Zif268-HIV binding sites + MET25 constitutive yeast promoter
NOT Gate Promoter Family Member (D001O1wt1)
Promoter Family Member (U097NUL+D050O22)
Promoter Family Member (U037O11+D050O22)
cI repressor from phage TP901 infecting L. lactis
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.
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
Promoter Family Member (U037O11+D062NUL)
A Double Reporter (LacZ-alpha and GFP-AAV) loaded on pSB3K5. For more information please refer to BBa_I732403.
Promoter Family Member (U085O11+D026O22)
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.
The puc promoter is the primary promoter for the puc operon. The puc operon consists of puc A and B coding regions responsible for the LH II antenna complex essential for light harvesting in Rhodobacter sphaeroides. The puc promoter is ultimately controlled by oxygen tension, and is a very strong promoter under low oxygen concentration.
PCR applified from plasmid pRKCBC3 provided by Dr. Neil Hunter.
Plac-rbs-rfp-Ter It produce rfp when there is enough IPTG or less lacI
Promoter Family Member (U073O11+D038O22)
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.
Promoter Family Member (U085O11+D062NUL)
POPS regulated and Lac inverter system with strong RBS
The biobricks BBa_Q04121 and BBa_B0034 where used to build this biobrick
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)
sgrS1+Terminator (small RNA regulator, conjugate part of ptsG1)
This part acts as a small regulatory RNA in E.coli.
critical region of SgrS/ptsG interaction
Pc+sgrS(wt)+Terminator (small RNA regulator, conjugate part of ptsG(wt))
This is the conjugate part of ptsG_wt.SgrS is the wild type small RNA regulator.
Critical region of SgrS(wt) complementary to ptsG(wt)
Placement of the operator sites was optimized for steric interference between the C2003 and RNA polymerase based on structural models.
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).
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.
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
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.
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
modified Lutz-Bujard LacO promoter,with alternative sigma factor σ38
Lac O and sigma 38 binding sites conserved sequences.
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.
Promoter Family Member (U085O11+D002O22)
Inverted tetR promoter with noncoding, spacer DNA upstream of it to make the total length 450 bp.
This part is in a series of inverted tetR promoters that have total lengths of 150, 250, 350, 450, 650, and 850 bp. We chose the region upstream of tetR in part Q04400 to be the noncoding DNA segment. This noncoding segment consists of the 3' end of tetR and B0015. None of the parts in this series was long enough to include the start codon of tetR, so a functional tetR protein should not be transcribed.
Primers were synthesized to bind to part Q04400, and then a PCR reaction was run.
NOT Gate Promoter Family Member (D001O11)
Inverted tetR promoter with noncoding, spacer DNA upstream of it to make the total length 350 bp.
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.
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
Promoter Family Member (U073O11+D002O22)
NOT Gate Promoter Family Member (D001O22)
Promoter Family Member with Hybrid Operator (D001O12)
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.
Placement of the operator site was determined to optimize steric interference between C2003 and RNA polymerase.
This promoter was rationally designed and synthesized.
Promoter Family Member (U049O11+D014O22)
Promoter Family Member (U073O26+D062O61)
Arsenic Promoter (ArsR regulated) with own RBS
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]
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
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.
Promoter Family Member (U049O11+D038O22)
endogenous promoter for Fig1 (Factor Induced Gene 1)
The Rec A(SOS) promoter has been PCR amplified from the chromosomal DNA from MG1655(Ecoli).
Double Promoter (LuxR/HSL, positive / cI, negative)
LasR and 3OC12HSL regulated promoter with a single Tet Operator (binding site for TetR repressor)
Promoter Family Member (U085O11+D062O22)
Comprised of araR binding sites and araE promoter. Regulates the expression of downstream genes with arabinose.
Forward primer used: GATCTG-GAATTCGCGGCCGCTTCTAGAG-CACAGCGTTCTTTGTAAG (Clamp sequence - Standard Biobrick prefix - first 18 base from the Biobrick) Reverse primer used: TGTGAC-ACTAGTA-GCCCTCCCGAATGTTGAG (Clamp sequence - SpeI site - last 18 base from the Biobrick)
Wild-type Bacillus subtilis 168 strain. 200 bp long bindingsite_promoter_bindingsite_RBS is amplified by PCR with dangling end primers with EcoRI-NotI-XbaI restriction site at 5??? and SpeI at 3??? and inserted into a Biobrick compatible vector. The sequence is taken from wildtype Bacillus subtilis
Inverted tetR promoter with noncoding, spacer DNA upstream of it to make the total length 650 bp.
Promoter Family Member (U097NUL+D002O22)
A Double Reporter (LacZ-alpha and GFP-AAV) loaded on pSB3C5. For more information please refer to BBa_I732401.
Lac system inverter controlling the expression of LasR
The biobricks BBa_Q04121, BBa_B0034 and BBa_K266002 where used to build this biobrick
CheZ is responsible for the dephosphorylation of flagellum-regulating protein CheY. It has been reported that cheZ-/- strain has a higher frequency of direction change and thus a narrower range of mobility. This device usually allows strong cheZ expression, while in the presense of IPTG, the transcription of cheZ is repressed by the overexpression of lambda cI repressor BBa_C0051.
cheZ CDS is cloned from Escherichia coli JM109 strain. Other sequences are from BioBrick parts.
The fusion protein omp(46-159)streptavidin(WT) with a 6-histidine tail is linked after a pTet promoter. The fusion protein coding region does not contain any terminator. The omp domain directs the expression of the fusion protein on the surface membrane
The pTet promoter, rbs are from the registry The fusion protein is self-designed
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.
LasR and 3OC12HSL regulated promoter with CI Operator 1 (binding site for lambda cI repressor)
Promoter Family Member (U073O11+D050O22)
Promoter Family Member (U097NUL+D026O22)
A Double Reporter (LacZ-alpha and GFP) loaded on pSB3C5. For more information please refer to BBa_I732403.
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 Family Member with Hybrid Operator (D001O47)
Double Promoter (cI, negative / LacI, negative)
to be used with transcription factor BBa_J34802
pcI (for positive control of pcI-lux hybrid promoter)
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
Promoter sequence with recognition site for ZntR transcriptional regulator protein.
NOT Gate Promoter Family Member (D001O33)
Promoter Family Member (U097O11+D050O22)
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.
Promoter Family Member with Hybrid Operator (D001O16)
SgrS2+Terminator (small RNA regulator, conjugate part of ptsG2)
This is the conjugate part of ptsG2-gfp.
Site specifically mutated for biological orthogonality.
critical region for SgrS/ptsG interaction
Promoter Family Member (U085O11+D038O22)
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.
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.
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.
consensus sequences based on [Jensen98].
This is identical to BBa_R0051 exept that some base has been modifed to alow for additional ZFP repressors to bind
PenP operator from Bacillus Lichenformis. Two operator sites are negatively regulated by PenI (C0074).
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)
promotor of the fet3 gene from S. cerevisiae, fet3 is repressed on high iron levels.
length of promoter was chosen to include binding sites and to obtain good primers
genomic upstream sequence of fet3 gene from the Saccharomyces Genome Database (http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=fet3)
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.
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.
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.
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.
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.
Constitutive promoter + 37℃ induced RBS + tetR + double terminator
we took advantage of the interaction between 37?? C induced RBS, tetR gene and pTet promoter to create the temperature regulating circuit. The following is a description of our prescribed strategy. First, we use the constitutive promoter, 37?? C induced RBS (K115002), the tetR gene and terminators. We will call this part the ???Regulator???. We speculate that when the ???Regulator??? and ???Production??? sections are combined, we can achieve a high bacteria growth rate and close to no target protein producing rate at T>37??C, and low bacteria growth rate and high target protein producing rate at T<37??C.
To regulate following gene by temperature
Promoter Family Member (U037O11+D002O22)
this is a modified version of the LacIQ promoter that should bind LacIQ tighter than the original.
Promoter Family Member (U097NUL+D038O22)
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
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)
Promoter Family Member (U037O11+D026O22)
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 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.
This is a mutant T7 promoter that includes a lacO sequence. This is repressible by the LacI protein and is induced in the presence of IPTG. The promoter sits in the 5' region of the gene of interest and initiates transcription when the cell expresses T7 RNA polymerase. Be sure you are using a cell strain that includes this gene (DE3).
There are three portions to the T7 promoter: the recognition site, the melting box (or TATA box) and the initation site. Mutations in these sequences caused differential behavior from the wild type.
The part consists of a sequences that resemble a T7 promoter and the lacO sequence found in E. coli.
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.
CMV promoter with inserted lac operator sites. Lac operator from POPI3Cat
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)
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 part produces GFP in the presence of sigma-70 E. coli RNA polymerase; however, the promoter contains two sites for a synthetic repressor to bind. Without the presence of this repressor, the part can serve as a strong constitutive promoter, as shown in the pictures below. For more information on the repressor, visit http://2015.igem.org/Team:Pitt/Protease/Project. The GFP produced is a mutant known as GFPmut3b, and the original citation is as follows: Cormack, B.P., Valdivia, R.H., and S. Falkow. FACS-optimized mutants of green fluorescent protein (GFP). Gene 173: 33-38 (1996). (http://www.sciencedirect.com/science/article/pii/0378111995006850) It has a maximum excitation at 501 nm and maximum emission at 511 nm. For more information, see part BBa_E0040.
The part containing GFP was cut with EcoRI and XbaI, and ligated with the annealed oligos to form the completed part. Note that due to the choice of technique, a Biobrick scar site remains between the promoter and RBS.
The RBS-GFP-terminator was obtained from as part BBa_E0840 from the 2015 Distribution (Plate 2, Well 24D). The pProt promoter was obtained as two short oligonucleotides annealed to form sticky ends. The part containing GFP was then cut with EcoRI and XbaI, and ligated with the annealed oligos to form the completed part.
Inverted tetR promoter with noncoding, spacer DNA downstream of it to make the total length 250 bp. The promoter faces the 5' direction.
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.
Promoter Family Member (U085O11+D014O22)
Promoter sequence with recognition site for ArsR transcriptional regulator protein. ArsR binds to the promoter sequence in the absence of As and releases on binding of As, therby activating transcription.
Promoter Family Member (U097NUL+D062O22)
NOT Gate Promoter Family Member (D001O44)
Promoter with standard -10 and -35 sites. A symmetric Zif23 dimer target site is after the -10 site.
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.
Promoter Family Member (U073O27+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
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.
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
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)
Promoter Family Member with Hybrid Operator (D001O26)
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).
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 is the promotor of yeast anb1 gene. Panb1 is a target of the rox1 repressor.
sequence and information from http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=SUC2
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).
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
Promoter Family Member (U061O11+D038O22)
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)
Promoter Family Member (U049O11+D062O22)
Promoter Family Member with Hybrid Operator (D001O24)
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.
A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000
Promoter Family Member (U097O11+D002O22)
Promoter Family Member with Hybrid Operator (D001O21)
Promoter Family Member (U097O11+D062O22)
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.
Double Promoter (constitutive / TetR, negative)
Promoter Family Member (U061O11+D026O22)
Double Promoter (cI, negative / TetR, negative)
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.
This is identical to BBa_I12007 exept that some base has been modifed to allow for additional ZFP repressors to bind.
NOT Gate Promoter Family Member (D001O66)
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
Constitutive promoter+37℃ induced RBS+tetR+double terminator+Ptet+RBS+GFP+double terminator
We want to utilize a temperature-regulated mechanism to control the expression period of a crystal protein. Therefore, we took advantage of the interaction between 37?? C induced RBS, tetR gene and pTet promoter to create the temperature regulating circuit. The following is a description of our prescribed strategy. First, we use the constitutive promoter, 37?? C induced RBS (K115002), the tetR gene and terminators. We will call this part the ???Regulator???.
Promoter Family Member (U097O11+D014O22)
The placement of the operator was designed to be near optimal for steric interference between C2003 and RNA polymerase.
Promoter Family Member (U061O11+D062NUL)
A Double Reporter (LacZ-alpha and GFP-AAV) loaded on pSB3K5. For more information please refer to BBa_I732401.
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).
Promoter of Tryptophan operon ( Tryptophan operon is an operon that codes for the components of tryptophan's synthesis)
2 SpeI enzyme restriction sites on Trp operon sequence.
Sequence of Trp operon was found at : http://biocyc.org/ECOLI/NEW-IMAGE?type=OPERON&object=TU00067 Part was added to the system as the upstream sequence of the forward primer in a PCR.
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.
Promoter Family Member (U073O26+D062NUL)
Promoter Family Member (U061O11+D014O22)
Promoter Family Member (U061O11+D050O22)
Promoter Family Member (U097O11+D062NUL)
PphoA promoter is a phosphate-responsive promoter. In high concentration of phosphate, it will be repressed while in low phosphate concentration, it will be activated.
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
NOT Gate Promoter Family Member (D001O55)
Double Promoter (LuxR/HSL, positive / P22 cII, negative)
Promoter from the lack operon cloned in the reverse orientation.
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.
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 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
tetR promoter with noncoding, spacer DNA upstream of it to make the total length 150 bp
This part is in a series of tetR promoter that have total lengths of 150, 250, 350, 450, 650, and 850 bp. We chose the region upstream of tetR in part Q04400 to be the noncoding DNA segment. This noncoding segment consists of the 3' end of tetR and B0015. None of the parts in this series was long enough to include the start codon of tetR, so a functional tetR protein should not be transcribed.
Promoter Family Member (U049O11+D026O22)
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.
Promoter Family Member (U049O11+D050O22)
Promoter Family Member (U061O11+D002O22)
The BBa_J23100 constitutive promoter with Lac systems inverter
The biobricks BBa_J23100 and BBa_Q04121 where used to build this biobrick
Promoter Family Member (U097O11+D026O22)
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
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
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
Promoter Family Member (U061O11+D062O22)
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.
Psuc2 promotor from yeast. Repressed by mig1
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.