This part holds the fluorescent protein mCherry attached to a degradation tag AAV which breaks down fluorescent protein over time.
We eliminated a restriction site from the gene.
This part was modified from a plasmid provided by the Murray lab at Caltech.
AID with CMV promoter and hGH-polyadenylation signal sequence
The BioBrick wtAID is an extended version of the existing AID BioBrick (BBa_K103001). It is built of 3 parts: CMV promoter, AID and hGH-polyA tail. AID: AID is known to be responsible for somatic hypermutation and the class-switch recombination of immunoglobulin in B cells. This enzyme of 28 kDa originally occurs in B cells but does also show activity after transfection into CHO cells. AID induces the deamination of cytidine to uridine at actively transcribed single strand DNA. The replacement of cytidine by uridine leads to a mismatch during DNA replication and integrates a single base substitution predominantly in the immunoglobulin genes. CMV promoter: CMV stands for cytomegalovirus. The CMV promoter is commonly used due to its very strong activity, and effectivity in a broad range of cell types. The BioBrick is therefore improved via addition of the strong promoter. hGH-polyA tail: The polyA tail consists of multiple adenosine monophosphates. hGH-polyA tail is a significant part for the translation and stability of mRNA. In eukaryotes, polyadenylation is part of the process that produces mature messenger RNA (mRNA) for translation. It, therefore, forms part of the larger process of gene expression.
It is a composite part consisting of 3 other Biobricks.
This is PBAD (BBa_I13453) mutant. -71 relative to the araC transcriptional start site was changed from A to G.
In order to regulate transcription from PBAD, araC binds three different sites called araO2, araI1, and araI2. In the absence of L-arabinose, araC binds both araO2 and araI1 and bends DNA preventing transcription from PBAD. In the presence of L-arabinose, however, araC has a conformational change because of L-arabinose and binds araI1 and araI2, enabling transcription from PBAD. Like the wild-type (I13453), this part does not have araO2 site which is essential for araC repressor function. Therefore, araC acts as an activator in the presence of L-arabinose.
This mutant was created by site-directed mutagenesis.
FadJ, gene for fatty acid degradation from E.coli str. K12
FadJ is the anaerobic counterpart of FadB. The protein is shown to have better degradation of medium and short length fatty acid together with FadI.
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 .
GFP and RFP linked with a flexible chain and a stop codon TAG is inserted in the flexible chain. This biobrick is under the control of T7 promoter and lac operator. This part is used to testify the function of PT7-TDRS (BBa_K567011) and tRNA(Asp)-TAG (BBa_K567013).
This sequence is the product of restriction cloning of the spacer2+RBS+mCherry downstream to the pLux promoter. In the presence of LuxR transcription can be induced by 3OC6HSL.
Cloning of the spacer2+RBS+mCherry downstream to the pLux promoter.
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 .
Parts I13007-I130014 are RBS test constructs for RBS characterization. Each consists of Lac or Tet promoter, one of the four RBS's B0030-B0033, a YFP coding protein, and B0015 terminator.
TIME DELAY 0.6:LuxI(RBS0.6)->LuxR->LuxPR->GFP
Sifan Wang, Ruosang Qiu, Shuqin Hu,Zhao Ma,Yunxin Long
This part contains part5 and part10. Arabinose will activate the promoter Pbad, which will cause production of protein LuxI and LuxR. The protein LuxI, LuxR and LuxPR make up the quorum sensing system, which leads to the expression of GFP.
We built this part by biobricks from the DNA distribution kit plates 2012.
This is PBAD (BBa_I13453) mutant. -60 relative to the araC transcriptional start site was changed from C to T.
''Escherichia coli'' osmoregulatory trehalose synthesis A (otsA)
Rashmi Sharma, Vishesh Jain, Kendrick Wang
RBS.3 (medium) -- derivative of BBa_0030
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
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
Long non-matching (scrambled) input strand to act as a negative control to the reporter formed by parts Part:BBa K779500 and Part:BBa K779502 or Part:BBa K779501 and Part:BBa K779502. Modifications: 5' IRD800 fluorophore, 3' phosphate. IDT DNA oligo order: /5IRD800/mAmC mUmAmA mCmCmU mAmAmC mUmAmC mAmAmC mAmAmC mAmCmU mAmUmC mCmA/3Phos/
In the domain, uses the same nucleotides as the Sa, but is scrambled so that the Sa and Sc domains are orthogonal. See more details at the MIT iGEM wiki: http://2012.igem.org/Team:MIT
The 434 cI repressor protein coding sequence is a 710 base-pair sequence with the standard RBS-compatible BioBrick prefix and the standard BioBrick suffix sections on its ends. It binds to the 434 regulatory sequence, BBa_R0052. The sequence contains a LVA tag for faster degredation and has no RBS.
References (unparsed) here: Nikolnikov,S., Posfai,G. and Sain,B. The construction of a versatile plasmid vector that allows direct selection of fragments cloned into six unique sites of the cI gene of coliphage 434. Gene 30 (1-3), 261-265 (1984) References (unparsed) here: Nikolnikov,S., Posfai,G. and Sain,B. The construction of a versatile plasmid vector that allows direct selection of fragments cloned into six unique sites of the cI gene of coliphage 434. Gene 30 (1-3), 261-265 (1984)
bhal laccase from Bacillus halodurans with T7 promoter, RBS and His-tag
bhal laccase from Bacillus halodurans with T7 and HIS
Construction Intermediate: mCherry, bacterial with RBS (B0034.J06504)
Combines BBa_B0034 RBS (Elowitz 1999) with BBa_J06504 mCherry, bacterial. This is an intermediate leading to BBa_J06702.
Niacin Chimeric GPCR with Fus1-EGFP reporter (2)
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
Protein generator converting TIPS to the protein cI from 434. Used as the input section for Quad Part Inverter Q01520.
SV40 nuclear localization sequence from SV40; yeast codon optimized
yeast codon-optimized nuclear localization sequence from simian virus 40
direct synthesis from published sequence
Inserting a BamHI/EcoRI fragment into the BglII/MfeI sites of J61015 destroys the restriction sites and introduces standard Biobrick flanking sequence. In the process, the OnRFP cassette is destroyed allowing Red to White screening of colonies.
PCR ca1007F/R on pSB1A2-J01064 (972 bp, EcoRI/SpeI) PCR ca1008F/ca1009R on pSB1A2-J01064 (972 bp, EcoRI/SpeI) PCR ca1007F/ca1009R on pSB1A2-J01064 (972 bp, EcoRI/SpeI) Sub into pSB1A2 (EcoRI/SpeI) Products are: Bca1007 NotI/BamHI transfer Bca1008 BamHI/EcoRI transfer Bca1009 NotI/EcoRI transfer ------------------------------------------------ ca1007F Forward EcoRI for 5' PspOMI NotI converter ctctggaattcgcggccgcttctagagggccctccctatcagtgatagagattg ca1007R Reverse SpeI for 3' BglII BamHI converter ccgctactagtagatcttataaacgcagaaaggcccac ca1008F Forward EcoRI for 5' BglII BamHI converter ctctggaattcgcggccgcttctagaagatcttccctatcagtgatagagattg ca1009R Reverse SpeI for 3' MfeI EcoRI converter ccgctactagtcaattgtataaacgcagaaaggcccac
Upstream construct of the synthetic multicellular bacteria
This part is a composite of BBa_K592016, BBa_K592006, and BBa_K592010 in that order. When the cells are exposed to blue-light, they will express amilGFP, a yellow chromoprotein. This protein causes the cells to have a strong yellow color.
BBa_K592016, BBa_K592006, and BBa_K592010 are available in the parts registry. They were obtained directly from the Uppsala-Sweden 2011 team.
TetR DNA binding domain containing BamHI site for protein fusions
Truncated version of tetracycline repressor TetR, consisting of 1 ??? 127 amino acids, containing DNA binding domain. The rest of the structure, 8-10 helixes responsible for tetR dimerization, were removed in order to keep tetR-DBD in monomer form and prevent from efficient DNA binding and transcription repression. In addition, we introduced BamHI site at C terminus for an easy in tetR-DBD frame fusions with other proteins. Thus, it can be used in two hybrid systems for both, homo and hetero dimerizations, also one can use these fusions to turn protein-protein interaction into the repression of transcription.
Deletion of 8-10 helixes might lower the stability of protein, also TetR-DBD is no longer responsive to anhydrotetracycline
This is the T7 Promoter which is activated when the T7 RNA Polymerase is produced. It provides very good PoPS (Polymerases Per Second) output, as well as it is highly orthogonal to E. Coli's natural transcription system. This one of the moderately active (Mutant D) variations we are using.
This gene codes for MotB, a part of the flagellar motor. MotA and MotB constitute the stator and are required for the flow of ions and torque generation.
* motA was amplified from genomic DNA of E. coli DH10B via PCR using the following primers: ** Fwd: GTTTCTTCGAATTCGCGGCCGCTTCTAGATGAAGAATCAAGCGCATCC ** Rev: GTTTCTTCCTGCAGCGGCCGCTACTAGTATCACCTCGGTTCGGCTGATGG
* The part was amplified from genomic DNA of E. coli str. K-12 substr. DH10B, complete genome (CP000948.1).
10x-Histidine (10x-His) Tag with double stop codon (TAATAA)
10x-Histidine tag with double stop codon TAATAA to allow for better extraction of tagged products and protein termination in a single part.
Constructed through oligonucleotide annealing
unidentified/uncharacterized CinR dependent
Synthesizes 3OC6HSL, which binds to LuxR. 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 HSL. This complex binds to a palindromic site on the promoter, increasing the rate of transcription.
An LVA tail (sequence: AANDENYALVA) was added to increase protein degradation. .
This is a reverse version of BBa_C0012, the lacIq repressor cds.
Standard biobrick prefix and suffix were used. Since PCR was used, DNA sequence verification was needed - no mutations were found.
PCR was used to amplify C0012 using primers that switched the biobrick prefix and suffix.
RBS.4 (weaker) -- derivative of BBa_0030
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
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
pLac promoter plus S. oneidensis tetraheme cytochrome c cymA
This part consists of the standard registry lac promoter (BBa_J33207) S. oneidensis tetraheme cytochrome c cymA (K917009).
pLac gene comes from E. coli BL21 genomic DNA cymA was cloned from S. oneidensis MR-1 genomic DNA
This part is used to screen the level of induction of the Psid promoter (I746364). The Psid promoter is induced by a class of phage activators (P2 ogr: I746350, PSP3pag: I746351, phiR73 delta: I746352).
The Psid promoter was put upstream of I13504.
registry (I13504) and own construct (I746364)
Arabinose regulated RBS-PhoA-His6tag-pNBEst13-Myctag-EstA
Marie Burghard, Jascha Diemer, Philipp Rottmann, Rene Sahm, Arne Wehling
This a a composite of 2 different BioBricks by an standard assembly, forming a scar in between. It contains the following units: AraC-AraO2-AraO1-AraPromo(PBAD)- RBS-PhoA-His6tag-pNBEst13-Myctag-EstA which is similiar to BBa_K808000 & BBa_K808030 Its a regulatory system, being induced by adding 1 to 2% of mw% L-Arabinose. It is made for regulating the cell surface Expression of our PET cleaving enzyme pNB-Est13, which is anchored c-terminal to EstA.
We were not able to add this part as an composite due to missing links and unedited registry pages. Necessary informations are featured in the sequence.
assembled GENEART synthesized product(BBa_K808030)and cloned regulatory unit (BBa_K808000), deriving from an expression system (pBAD-vector)
Contains both a BbsI and a BstXI restriction site. When cut with both enzymes, it leaves an EcoRI and a PstI site.
70 bp from Eco to Spe or Xba to Pst, to allow for gel extraction. The middle portion is largely noise, intended as filler to raise the size of the part.
Spider Silk 1x Subunit "W" with Met (ATG) start codon
Spider silk subunit native sequence; contains two elasticity domains and one strength domain, contains added Met (ATG) on 5??? end.
Created using mutagenesis PCR to add upstream Met (ATG) to part BBa_K844002
Sensory rhodopsin I (SRI) with HtrII & Tar, sensitive to orange light
Sensory rhodopsin I fusion with HtrII & Tar, which is sensitive to orange light, with sensing spectra covering from 590-650 nm. We used it for sensing blur-green light to achieve phototaxis and switch for downstream gene expression.
Pfam (version 26.0) was used to predict the domain of fusion protein. Linker was refereed and modified from previous research study of SRI fusion with HtrII.
From genomic sequence of bacterial N. pharaonis (DSM 2160) and E.coli K12.
Population 2 for use with the dual strain toggle
xylR+PxylA+RBS+GFP+Double Terminator
Sensory rhodopsin II (SRII) with HtrII & Tar, sensitive to blue-green light
Sensory rhodopsin II fusion with HtrII & Tar, which is sensitive to blue-green light, with sensing spectra covering from 400-500 nm. We used it for sensing blur-green light to achieve phototaxis and switch for downstream gene expression.
Pfam (version 26.0) was used to predict the domain of fusion protein. Linker was refereed and modified from previous research study of SRII fusion with HtrII.
tthl (Laccase from Thermus thermophilus) coding sequence
cI repressor from E. coli phage lambda (+LVA)
Vinay S Mahajan, Brian Chow, Peter Carr, Voichita Marinescu and Alexander D. Wissner-Gross
Coding region for the cI repressor based on cI repressor from bacteriophage lambda modified with an LVA tail for rapid degradation of the protein. cI repressor binds to the cI regulator (BBa_R0051).
References (unparsed) here: A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000 Octamerization of CI repressor is needed for effective repression of PRM and efficient switching from lysogeny. Ian B. Dodd,1 Alison J. Perkins, Daniel Tsemitsidis, and J. Barry Egan , Genes and Development (Vol 15, No. 22) 3013-3022: 2001 References (unparsed) here: A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000 Octamerization of CI repressor is needed for effective repression of PRM and efficient switching from lysogeny. Ian B. Dodd,1 Alison J. Perkins, Daniel Tsemitsidis, and J. Barry Egan , Genes and Development (Vol 15, No. 22) 3013-3022: 2001 BBa_C0051 cI repressor is based on the cI repressor from the Elowitz's repressilator. It has been modified to include a rapid degradation LAA tail, and includes the BioBrick standard assembly head and tail restriction sites. The RBS has been removed. The stop codon has been changed from TAA to a double stop codon TAATAA.
Elowitz, M. B. Transport, Assembly, and Dynamics in Systems of Interacting Proteins. Thesis, Princeton Univ., Princeton (1999).
RI7-Olfr154 Isoamylacetate chimeric GPCR
BBa_K775001 encodes a chimeric isoamylacetate-sensing G-protein coupled receptor which can be functionally expressed in S. cerevisiae. This chimeric receptor is composed of the ligand-binding domain of the mouse Olfr154, flanked by the N- and C- terminals of the rat OR RI7.
N-terminus: Olfactory receptor-like protein I7 (olr226), Rattus norvegicus (Rat) Ligand binding domain: Olfr154 NM_013728, Mus musculus, (mouse) C-terminus: Olfactory receptor-like protein I7 (olr226), Rattus norvegicus (Rat)
enhanced yellow fluorescent protein derived from A. victoria GFP
Leah Ferrell, Sanket Shah, Shayan Ghassemi, Zakaraya Ashour
pLasR is off by default and can be actated by LasR. LuxI converts SAM to AHL.
This is PBAD (BBa_I13453) mutant. -59 relative to the araC transcriptional start site was changed from C to T. Also, C at -48 was changed to T.
This mutant was created by random mutagenesis.
Overlap extension of synthetic oligonucleotides
In complex with HSL, LuxR binds to the Lux promoter, activating transcription from Pr <bb_part>BBa_R0062</bb_part>, and repressing transcription from Pl <bb_part>BBa_R0063</bb_part>. 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 activator, LuxR complexed to HSL. 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.
2 silent point mutants were introduced in the coding sequence to remove internal XbaI and PstI sites. Mutation sites were chosen to replace codons commonly used in E. coli with codons used at a similar frequency.
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.
Spider Silk 1x Subunit "B" (Balanced tRNA codon optimized) with Met (ATG) added
Spider silk subunit optimized to use a reduced set of tRNA codons (1 or 2 codons per amino acid); contains two elasticity domains and one strength domain, also contains 5??? Met (ATG) codon.
Created using mutagenesis PCR to add upstream Met (ATG) to part BBa_K844004
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 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.
modified AID with CMV, hGH-polyA and eGFP
The BioBrick wtAID is an extended version of the existing AID BioBrick (BBa_K929001). It is built of 3 parts: CMV promoter (BBa_I712004), superAID, eGFP (BBa_K404316) and hGH polyadenylation signal sequence (BBa_K404108). AID: AID is known to be responsible for somatic hypermutation and the class-switch recombination of immunoglobulin in B cells. This enzyme of 28 kDa originally occurs in B cells but does also show activity after transfection into CHO cells. AID induces the deamination of cytidine to uridine at actively transcribed single strand DNA. The replacement of cytidine by uridine leads to a mismatch during DNA replication and integrates a single base substitution predominantly in the immunoglobulin genes. The AID motif is naturally terminated with the Nuclear Export Sequence (NES) that causes the protein to translocate from the nucleus to the cytoplasm. Additionally, upstream, dysfunctional Nuclear Localization Sequence (NLS) is located. Due to the fact that AID mutates the actively transcribed single stranded DNA, it is supposed that the direction of the enzyme to the inside of the nucleus would improve the mutation rate. That is why we called it superAID Functional NLS sequence: This part of the BioBrick directs the expressed protein into the nucleus, where it can mutate stronger. Kozak sequence Kozak consensus sequence is added upstream of the AID mutant to express the protein stronger. CMV promoter: CMV stands for cytomegalovirus. The CMV promoter is commonly used due to its very strong activity, and effectivity in a broad range of cell types. The BioBrick is therefore improved via addition of the strong promoter. hGH polyadenylation signal sequence: Polyadenylation is a significant part for the translation and stability of mRNA. In eukaryotes, it is part of the process that produces mature messenger RNA (mRNA) for translation. It, therefore, forms part of the larger process of gene expression. hGH terminator gives a signal to start polyadenylation in the translation process. eGFP: In order to test where and if the AID modified sequence is functional we added to it a eGFP protein. It is used as a marker gene for detection of transfected cells, e.g. tumor cells.
4 parts: CMV promoter (BBa_I712004), superAID(BBa_K929001), eGFP (BBa_K404316) and hGH polyadenylation signal sequence (BBa_K404108)
tctA_503: large subunit A1 of the tripartite tricarboxylate transporter family
Valentina Herbring, Sebastian Palluk, Andreas Schmidt
mRFP (DsRed) derived, altered to be a biobrick by removing a PstI site and adding in the ends.
Made a point mutation to eliminate a PstI site in the middle and then added BioBrick ends using PCR. Sequenced using primers that bind to the pSB1A2 plasmid on either side of the brick.
mCherry from Roger Y. Tsien's lab, altered to be BioBrick compatible
Promoter can be inserted into the adapter containing BglII and BamHI sites. The activity of inserted promoter can be quantificationally measured using ONPG assay or fluorescent assay.
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 lambda (cI regulated) with RFP reporter
Promoter lambda - cI regulated - (R0051) with RFP (E1010) as a reporter. It can be used as a detector of the glucose metabolism.
Riboswitch that is highly sensitive to the F- ion. We have characterized this part in three different chassis: TOP10 e.coli, Laboratory strain bacillus subtilis and a strain of bacillus subtilis with its normal fluoride riboswitch knocked out (kindly provided by the Breaker lab in Yale). Results of Miller assays for these three chassis are shown below.
Because this is a positive regulator (it ceases transcriptional termination in the presence of F- ions), it was decided that it should be placed upstream of a direct reporter, in this case lacZ as our reporter.
This is PBAD (BBa_I13453) mutant. -69 relative to the araC transcriptional start site was changed from C to A.
This part accepts POPS to produce ECFP under the medium RBS B0032
Different strength of green fluorescence will be generated determined by the concentration of IPTG.
This sequence is the product of restriction cloning of the theophylline riboswitch downstream to the pLux promoter. In the presence of LuxR transcription can be induced by 3OC6HSL.
Cloning of theophylline riboswitch downstream to the pLux promoter
Reverse RBS (RBSrev) -- corresponds to BBa_B0030
Repeats in oligos caused unusual products during cloning
The sequence comes from Ralstonia eutropha H16 adapted to become a standard BioBrick.
Phosphoacetylglucosamine mutase (N-acetylglucosamine-phosphate mutase); enzyme of UDP-N-acetylglucosamine (UDP-GlcNAc) biosynthesis (1) This enzyme is part of the pathway to make the sugar subunits that make up the chitin polymer. This pathway was taken from yeast but has been shown to function in Acetobacter xylinum.
Gibson assembly fragment must have ovelapping sequences of about 50 bp.
The sequence is from the candida genome database orf19.5013 (C. albicans SC5314. It contains a ribosome binding site. It was synthesized in <500bp pieces for Gibson Assembly and assembeled into pSB1C3.
Jay Blumling, Debra Lin, Madeleine Sheldon-dante, Fred Tan, MIT SMUG
PC and AraC from the AraBAD operator. PC and AraC are on the complementary strand, reading right to left.
Positively regulated, OmpR-controlled promoter from OmpC gene (R0082) directing transcription of mRFP (I13507). Constructed with standard assembly method except R0082 was isolated using VF and VR-directed PCR. Consequently, there may be silent mutations that were inadvertently introduced. An alternative clone is M30012. Cells bearing this construct are pale pink.
Tetracycline repressor DNA binding domain fusion with truncated version of UVR8 (tetR-DBD-UVR8). UVR8 is a UV-B sensing receptor in plants necessary for UV-B protection. In dark state UVR8 forms a dimer which is monomerized by the UV-B light (280-315 nm). A truncated version of UVR8 (14-440 amino acids) was fused with tetR DNA binding domain (tetR-DBD), lacking of dimerization domain. Such mutant shows no repression of Ptet promoter, however fusion with dimerizing protein (UVR8) returns tetR-DBD activity. Later UV-B exposure breaks UVR8 dimer and releases tetR-DBD-UVR8 from Ptet, activating transcription. Thus, this tetR-DBD-UVR8 can be used as a one step UV-B on switch.
Contains two illegal PstI sites in UVR8 coding region and BamHI site was used for protein fusion
tetR-DBD was cloned from E.coli tetracyclin repressor tetR from transposon Tn10 cDNA of UVR8 was provided by Roman Ulm, University of Geneva.
3-Hydroxy-3-Methyl-Glutaryl-CoA-Reductase from Yeast
This is a truncated version of the 3-Hydroxy-3-Methyl-Glutaryl-Coenzyme-A-Reductase (HMG-CoA-R) from ''Saccharomyces cerevisiae''. It catalyses the reaction of 3-Hydroxy-3-Methyl-Glutaryle-Coenzyme-A to Mevalonate which occurs in the Mevalonate Pathway to Isopentylpyrophosphate which is the Precurser of all Isoprenoides. The enzyme is naturally a membrane bound enzyme. The membrane anchor is situated N-terminal. This region is also essential for the degradational control of the enzyme. By deleting the N-terminal part to Aminoacid (inclusive) the enzyme is expected to be water soluble and deregulated.
The truncation was done according to the following publication: Rico, Pardo, Orejas: 'Enhanced Production of a Plant Monoterpene by Overexpression of the 3-Hydroxy-3-Methylglutaryl Conenzyme A Reductase Catalytic Domain in Saccharomyces cerevisiae'; Applied and Enviromental Microbiology; Vol 76 p. 6449-6454; 2010
The original source of this part is the genome of Saccharomyces cerevisiae. We obtained the sequence by de novo synthesis. For that we used data from the Saccharomyes Genome Database. We inserted some calm mutations to avoid conflicts with the Cloning Standard 10.
This part carries the gene for a proteorhodopsin pump, which can generate a proton motive force for making ATP.
We codon optimized the gene for E. coli and eliminated four restriction sites.
This is a construct carrying the sfGFP gene with low-efficiency RBS, under lac operator control. lac repressor (encoded from lacI gene) is the regulator protein: in absence of lactose the protein binds to lac operator, preventing RNA-polymerase binding and transcription.Low, constitutive levels of transcription can be seen anyhow.
This construct has ben obtained from ligation between BBa_K731500 and BBa_E0240.
The Isaacs key1 riboregulator taR12 is modeled on the hok/sok post-segregational killing method of plasmid R1. It makes 25 base pairs with its counterpart, the riboregulator J01010 (aka "lock 1" or "crR12"). Isaacs documentation (see reference #1): Upon induction of the araC promoter (using 0.25% arabinose solution), the taR12 key activated the corresponding crR12 lock to express the downstream indicator, GFP, to levels of fluorescence 19-fold greater than without taR12 expression. Maximal expression was reached at 70 minutes after addition of arabinose solution, and was maintained after that point.
Binding region for LasR protein (positive regulation)
"Analysis of the Pseudomonas aeruginosa Elastase (lasB) Regulatory Region". Lynn Rust, Everett Pesci, and Barbara Iglewski
This is PBAD (BBa_I13453) mutant. -58 relative to the araC transcriptional start site was changed from A to C.
engineered cyan fluorescent protein derived from A. victoria GFP
Quadpart Amplifier (B0034.C0062.B0015.R0062)
This is PBAD (BBa_I13453) mutant. -70 relative to the araC transcriptional start site was changed from G to A.
PLac activation induces the expression of the Lactose permase (Lac Y) gene, LacI repressor protein with GFP (+LVA tag) as a reporter.
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
This part is a direct fusion of the theophylline riboswitch (BBa_K784005) and the mCherry protein (BBa_J06504) which was amplified from BBa_J06702. The part was generated by [http://openwetware.org/wiki/Assembly_pcr Assembly_pcr]. This part was to be used in order to characterize the effect of theophylline concentration on the level of translation of the mCherry protein.
The theophylline riboswitch had to be directly fused to the start codon of the mCherry protein. To achieve this, [http://openwetware.org/wiki/Assembly_pcr Assembly_pcr] was used.
This part is a composite part of BBa_K784005 and BBa_J06504
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)
In order to regulate transcription from PBAD, araC binds three different sites called araO2, araI1, and araI2. In the absence of L-arabinose, araC binds both araO2 and araI1 and bends DNA preventing transcription from PBAD. In the presence of L-arabinose, however, araC has a conformational change because of L-arabinose and binds araI1 and araI2, enabling transcription from PBAD. Like the wild-type (I13453), this part lacks araO2 site which is essential for araC repressor function, araC acts as an activator in the presence of L-arabinose.
Grace Kenney, Daniel Shen, Neelaksh Varshney, Samantha Sutton
Coding region for the LacI protein with an LVA degradation tail and without an RBS. LacI binds to the pLac regulator <bb_part>BBa_R0010</bb_part> and PLlac01 hybrid regulator <bb_part>BBa_R0011</bb_part> and inhibits transcription. IPTG (Isopropylthiogalactoside) binds to LacI and inhibits its operation, therefore promoting transcription. A rapid degredation tail (LVA) has been added to improve the High to Low performance of this part.
References (unparsed) here: Elowitz, M.B., Leibler, S. A synthetic oscillatory network of transcriptional regulators. Nature 403, 335-338 (2000). [ELOW00] References (unparsed) here: Elowitz, M.B., Leibler, S. A synthetic oscillatory network of transcriptional regulators. Nature 403, 335-338 (2000). [ELOW00] Sequence taken from the repressilator of Elowitz and Leibler (2000). The obtained sequence was compared to the wild-type sequence for LacI obtained through a database search. The sequence had been modified from the wild-type in that wild-type GTG start was changed to an ATG start (note, actual ORF in E.coli has several GTG starts it would seem). The LVA tag has been added for quicker degradation. Incompatible with systems containing LacI, lactose, or IPTG.
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.
"It contains a constitutive promoter, GvpC protein , GFP protein and a terminator. This part can be used as a control of (J23106+P0440+R0040+B0034+GVPC+E0840). It can express GvpC protein and GFP constantly. When it transformed with (J23106+P0412+R0011+B0034+GVPA+E0840), the bacteria may can float with GFP!"
This part can be used as a control of (J23106+P0440+R0040+B0034+GVPC+E0840). It can express GvpC protein and GFP constantly. When it transformed with (J23106+P0412+R0011+B0034+GVPA+E0840), the bacteria may can float with GFP.
Promoter(Pbad)-lux operon-DT + lumP(K769019)
R6K origin of replication requires a pir+ or pir116 strain for replication in the absence of a second origin. In most E. coli strains, this is a silent feature. {pSB1A2-Bca1011}
PCR ca1011F/R on pG80ko (445 bp, EcoRI/SpeI) Sub into pSB1A2-I13522 (EcoRI/SpeI) Product is Bca1011 ---- ca1011F Forward Biobricking of R6K GGACTgaattcgcggccgcttctagagtgattcgcacgggcccatg ca1011R Reverse biobricking of R6K ccgctactagtaGCAGTTCAACCTGTTGATAG
araC-Pbad regulated XylE - catechol 2,3-dioxygenase from P.putida with terminator
Tet operator with CFP reporter (with LVA tag) [R/Tc+]
Christopher Batten, Victoria Chou, Kenneth Nesmith
This is PBAD (BBa_I13453) mutant. -66 relative to the araC transcriptional start site was changed from T to G.
enhanced cyan fluorescent protein derived from A. victoria GFP
June Rhee, Connie Tao, Ty Thomson, Louis Waldman
Cyan fluorescent protein (ECFP) reporter coding sequence without the Ribosome Binding Site. Modified with an LVA tail for rapid degradation of the protein and faster fall time for the emission.
BBa_E0022 cyan fluorescent protein is based on BioBrick part BBa_E0021. It has been modified to include a rapid degradation LVA tail, and includes the BioBrick standard assembly head and tail restriction sites. The RBS has been removed. The stop codon has been changed from TAA to a double stop codon TAATAA.
Modified from <bb_part>BBa_E0021</bb_part>.
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.
Luminesensor repressible LacIM generator
LacI mutant driven by luminesensor repressible SulA promoter in 408 form.
B0034, CDS of LacI mutant and B0015 were connected directly to SulA promoter in 408 form.
Sequene of the LacI mutant is from Partregistry.
AroY: Catalyzes protocatechuate to catechol
Sascha Hein, Sven Rumpf, Daniel Sachs, Arne Wehling, Philipp Rottmann
Eukaryotic PoPs -> IaaH. Indoleacetamide hydrolyase for Auxin generator, with kozak
This part contains the coding sequence for Indoleacetamide hydrolyase preceded by a Kozak sequence, for expression in eukaryotic plasmids.
Kozak is included, as it cannot be added by biobrick. This part can be used to assemble biobricks for integration onto Xenopus tropicalis genome. It can also be used directly by injection into a fertilised egg, using the ready to use Xenopus plasmid BBa_K812000.
This part was created from BBa_K515100, itself from P. savastanoi (By Imperial college)
This part is involved in TAT protein export pathway of E.coli. The part consist of three genes expressing tatA,B,C proteins. They are the components of Tat complex on cell membrane, where protein with torA signal can go through
This parts have very long region so that we have to divide this parts into two regions to read sequence.
They are derived from tatABCD operon in Escherichia coli???s genome.
K206000+B0034+pelB+Cutinase+Polyhistidine Tag
Full Cutinase Construct on psB1A3 backbone.
There's no terminator so it needs to be used with a backbone that ends with a 3
This part generates LasI(which generate 3OC12HSL) in the presence of 3OC6HSL. We constructed it with (???) and (Plux,LasI part name).
We make this part in the middle of the progress of making the part plac-RBS-cI-T-pCon(0.856)-RBS-crogfp-T-pRM-anticro-T.
The expression of YFP and tetR is under the control of microaerobic promoter vgb. The expression of tetR will repress Ptet.
Because vgb promoter induces high expression under microaerobic conditions and induces lower expression under anaerobic conditions, we used medium RBS to resist it from inducing expression under anaerobic conditions.
vgb promoter comes from Vitreoscilla sp.
This biobrick is constructed by putting modified enzyme luciferase under promoter T7 and controlled by lac operator. 2 AGG codons and 2 GCG codons are inserted after the ATG start codon of wild type luciferase (BBa_I712019). Modified luciferase keeps the activity of converting luciferin into oxyluciferin, during which bioluminescence will emit. This part is one of the reporter genes to testify the influence of different number of rare codons in regulating protein biosynthesis. This part is used as a measurement to testify the function of LacI -Ptac-tRNA(Arg) (BBa_K567001) or sulA promoter-tRNA(Arg) (BBa_K567002). Cell is cultured in 50ug/ml kanamycin 10ug/ml tetracycline LB liquid medium. When the OD600 of the culture reaches 0.3 IPTG is added to make the final concentration 0.5nM to induce the synthesis of tRNA. Ultrasonication is used to release the luciferase from the cell. Sonics ON 3 seconds, OFF 3 seconds, total ultrasonication time 3minutes. Amount of bioluminescence produced can be detected using luminometer.
PCR is used to insert the 2 AGG codons into the gene after ATG.
T7 promoter is derived from pET28A. Wild type Luciferase from BBa_I712019.
Test mechanism for the effectiveness of TetR in repressing pTet. Also used to show the effect of ATc on TetR
Built by Josh as an intermediate in screening plasmid construction.
COMT (caffeic acid-O-methyl transferase) with ribosome binding site. Enzyme methylates -OH on C4 of the caffeic acid aromatic ring to produce ferulic acid. COMT is the third gene in the vanillin biosynthesis pathway,
Used COMT cDNA as genomic DNA contains introns
Genes and Enzymes Involved in Caffeic Acid Biosynthesis in the Actinomycete Saccharothrix esanaensis, Berner M, et al, Journal of Bacteriology 188(7): 2666, (2006) Gowri G, et al, Molecular cloning and expression of alfalfa S-adenosyl-L-methionine: caffeic acid 3-0-methyltransferase, a key enzyme of lignin biosynthesis, Plant Physiology 97(1):7 (1991) Downregulation of Caffeic Acid 3-O-Methyltransferase and Caffeoyl CoA 3-O-Methyltransferase in Transgenic Alfalfa Impacts on Lignin Structure and Implications for the Biosynthesis of G and S Lignin, Guo D, et al, Plant Cell. January; 13(1): 73 (2001)
mCFP with kozak sequence for eukaryotic expression
mCFP fused with a kozak sequence to enhance its expression in eukaryotic cells. To be used with an eukaryotic expression system such as pSC2+ (K812000) and related parts.
PCR and cloned in pSB1C3 using EcoRI and PstI
PCR amplified with kozak sequence from DNA coming from Alfonso Jaramillo's lab
This protein domain can only be used within the TAL-effector-toolkit designed by the IGEM2012 Team Freiburg. The codon usage was modified to reduce homology in the sequence. A silent point mutation was inserted into the BsmBI restriction site in the chloramphenicol gene.
This Part was synthesized and flanked with BsmBI-restriction sites.
Inducible araC-pBAD promoter regulating WT CysE
The CysE gene (K731000) is here regulated by the araC-pBAD promoter (K731200), which is inducible by arabinose.
CysE was inserted after araC-pBAD in his vector, cutting the former with X&P and the latter with S&P.
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
TIME DELAY0.01:LuxI(RBS0.01)->LuxR->LuxPR->GFP
Sifan Wang,Ruosang Qiu,Jianzhao Chi,Muxin Yu
This part contains part8 and part10. Arabinose will activate the promoter Pbad, which will cause production of protein LuxI and LuxR. The protein LuxI, LuxR and LuxPR make up the quorum sensing system, which leads to the expression of GFP.
p-ABA overproduction composite without promoter
PABA is derived from the aromatic branch point precursor chorismate in two steps. Aminodeoxychorismate (ADC) synthase converts chorismate and glutamine to ADC and glutamate. The conversion of chorismate and glutamine to ADC and glutamate determines the rate and therefore overexpression of ADC synthase leads to overproduction of pABA. ADC synthase is composed of two subunits, PabA and PabB. Finally ADC lyase removes pyruvate from ADC, aromatizes the ring, and generates PABA.
pabB and pabA constitute an operon that can be controled by any promoter.
This is the T7 Promoter which is activated when the T7 RNA Polymerase is produced. It provides very good PoPS (Polymerases Per Second) output, as well as it is highly orthogonal to E. Coli's natural transcription system. This one of the moderately active (Mutant E) variations we are using.
RBS+ispS is same BBa_K649400. PlacIQ was used the part BBa_.
RBS+ispS : GeneArt Product PlacIQ : Part(BBa_)
galK is the leader sequence of galactokinase (GalK)
galK is the leader sequence of galactokinase (GalK),and Spot42 sRNA controls the synthesis of the galactokinase (GalK) in response to the availability of glucose in the environment,by blocking the recognition of antiSD motif to RBS.The repression is modest(about 2.6 fold),maybe there is no subsequent degradation caused by the major degradosome,RNaesE and RNaseIII. Note:The ClaI site is blocked by GATC methylation due to a mistake.It should be amplified by PCR to get rid of dam methylation before ClaI degestion
Reference: [1] Johannes H. Urban and Jo?? rg Vogel, Translational control and target recognition by Escherichia coli small RNAs in vivo, Nucleic Acids Research, 2007, Vol. 35, No. 3 [2] Boris G??rke and J??rg Vogel, Noncoding RNA control of the making and breaking of sugars, GENES & DEVELOPMENT 22:2914???2925 ,2008
PenP operator from Bacillus Lichenformis. Two operator sites are negatively regulated by PenI (C0074).
UDP-N-acetylglucosamine pyrophosphorylase, catalyzes biosynthesis of UDP-N-acetylglucosamine from UTP and N-acetylglucosamine 1-phosphate; functional homolog of S. cerevisiae Qri1p; alkaline upregulated. This gene is part of the pathway that synthesizes the sugar subunit of the polymer chitin.
Part was synthesized from <500bp overlapping Gibson Assembly fragments
candida genome database orf19.4265 (C. albicans SC5314)
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.
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).
autoinducer inactivation enzyme from Bacillus; hydrolyzes acetyl homoserine lactone
Vinay S Mahajan, Brian Chow, Peter Carr, Voichita D. Marinescu and Alexander D. Wissner-Gross
Coding region for the autoinducer inactivation enzyme A (aiiA) LVA tagged. The gene was originally isolated from Bacillus sp. 240B1 and it encodes an enzyme that catalyzes the degradation of N-acyl homoserine lactones (AHLs)--quorum sensing autoinducers.
References (unparsed) here: Dong,Y.H., Xu,J.L., Li,X.Z. and Zhang,L.H.: AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora, Proc. Natl. Acad. Sci. U.S.A. 97 (7), 3526-3531 (2000). http://www.pnas.org/cgi/content/full/97/7/3526 Lee SJ, Park SY, Lee JJ, Yum DY, Koo BT, Lee JK.: Genes encoding the N-acyl homoserine lactone-degrading enzyme are widespread in many subspecies of Bacillus thuringiensis, Appl Environ Microbiol 2002 Aug;68(8):3919-24. http://aem.asm.org/cgi/content/full/68/8/3919?view=full&pmid=12147491 References (unparsed) here: Dong,Y.H., Xu,J.L., Li,X.Z. and Zhang,L.H.: AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora, Proc. Natl. Acad. Sci. U.S.A. 97 (7), 3526-3531 (2000). http://www.pnas.org/cgi/content/full/97/7/3526 Lee SJ, Park SY, Lee JJ, Yum DY, Koo BT, Lee JK.: Genes encoding the N-acyl homoserine lactone-degrading enzyme are widespread in many subspecies of Bacillus thuringiensis, Appl Environ Microbiol 2002 Aug;68(8):3919-24. http://aem.asm.org/cgi/content/full/68/8/3919?view=full&pmid=12147491 BBa_C0060 insert contains open reading frame (nucleotides 49-801) of the GeneBank sequence AF196486 followed by the LVA tag and two double stop codons inserted in the BioBrick prefix and suffix flanking regions. The original stop codon was TAG and in the present sequence it was substituted by TAATAA.
AF196486 from Bacillus sp. 240B1 putative metallohydrolase (aiiA) gene. Dong,Y.H., Xu,J.L., Li,X.Z. and Zhang,L.H.: AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora, Proc. Natl. Acad. Sci. U.S.A. 97 (7), 3526-3531 (2000).
This is PBAD (BBa_I13453) mutant. -71 relative to the araC transcriptional start site was changed from A to G. Also, C at -22 was changed to T
3,7-dimethylxanthine N-methyltransferase CaDXMT1-strep
3,7-dimethylxanthine N-methyltransferase CaDXMT1 from ''Coffea arabica'' followed by a strep-tag for purification and/or detection via western blot.
http://www.ncbi.nlm.nih.gov/nuccore/AB084125 synthesized by IDT, yeast codon optimized
RhlR can sense N-butyryl-HSL in the media and can activate transcription from R0071. It can be used as part of a receiver device.
Long reporter top strand with bulge MammoBlock
Reporter top strand with a 4nt bulge. Modifications: 5' RQ quencher, 3' Alexa florophore, all bases modified with 2'O Methyl (ordered as a DNA oligo with Us instead of Ts). IDT DNA oligo order: /5IAbRQ/mCmA mCmCmC mAmCmU mCmCmC mAmCmU mUmCmU mCmCmC mAmCmC mA/3AlexF488N/
Sequence designed to be orthogonal to the HEK293 transcriptome, and to have a high melting temperature. 4-nucleotide bulge added 10-nt from the 3' end as a potential protection mechanism from cleavage by RISC as suggested by Iba et al. (2011). See more details at the MIT iGEM wiki: http://2012.igem.org/Team:MIT
fhlA-E363G (engineered trascriptional activator fhlA with a mutation)
A to G (different from E. coli K-12 MG1655)
Contains an IPTG inducible promoter an RBS, RFP, and terminator.
There are two transcriptional factors which regulates the ADH2 promoter: ADR1 a carbon source-responsibe zinc-finger2 and CAT83. In the absence of glucose both cis-acting sites in the promoter are bound cooperatively by the transcriptional activators. ADR1 binds to the UAS1 site while CAT8 binds to the UAS2/CSRE site and regulates positively the transcription of YAP1 protein by the activation of the ADH2 promoter. The presence of glucose downregulates the levels of the transcription factors (ADR1 and CAT8) which results in the depletion of the production of YAP1 protein by the repression of the ADH2 promoter.
The sequences of the promoter and the gene which encodes the YAP1 protein comes from yeast Sacharomyces cerevisiae genome.
tctA_505: large subunit A2 of the tripartite tricarboxylate transporter family
Colicin E1 operon of E. coli expressed under a constitutive promoter (BBa_J23119).
BBa_K150007 was not available from the registry, so its sequence was cloned from K150009 by PCR.
OriT-R (Origin of Transfer for the R751-plasmid nic region)
OriT-R751 (Origin of Transfer for the R751-type conjugative plasmid) is the nic region, where the relaxosome nicks the plasmid and conjugative transfer by R751 plasmid machinery begins via rolling circle replication.
Enterobacter aerogenes plasmid R751 NCBI:NC_001735
This is the LC Cutinase gene used to break PET into terephthalic acid and ethylene glycol.
Eliminated BioBrick restriction sites and added a TAA double stop.
Modified from the paper Isolation of a Novel Cutinase Homolog with Polyethylene Terephthalate-Degrading Activity from Leaf-Branch Compost by Using a Metagenomic Approach by Sulaiman et al.
7-methylxanthine N-methyltransferase CaMXMT1-strep
7-methylxanthine N-methyltransferase CaMXMT1 from ''Coffea arabica'' followed by a strep-tag for purification and/or detection via western blot.
Synthesized by IDT, Yeast codon optimized
TIME DELAY1.0:LuxI(RBS1.0)->LuxR->LuxPR->GFP
Sifan Wang,Ruosang Qiu,Xinyi Yao,Qingshu Wu
This part contains BBa-K750001 and BBa_K750007. Arabinose will activate the promoter Pbad, which will cause production of protein LuxI and LuxR. The protein LuxI, LuxR and LuxPR make up the quorum sensing system, which leads to the expression of GFP.
ECFP Coding Device with Promoter, RBS, Coding Sequence, and Terminator
IPTG Inducible Promoter, RBS, ECFP with LVA tag, Terminator
bifunctional Cyclase from Gibberella fujikuroi
Genomic sequence of Gibberella fujikuroi.
This is PBAD (BBa_I13453) mutant. -56 relative to the araC transcriptional start site was changed from A to G.
ArgR binding sites (including 16 binding sites)
This is PBAD (BBa_I13453) mutant. -62 relative to the araC transcriptional start site was changed from A to G.
This is PBAD (BBa_I13453) mutant. -63(T to C), -42 (C to A), and +9 (T to A) relative to the araC transcriptional start site were changed.
Here, pCon(J23114) constantly expressing the fusion protein crogfp at 10% of the strength of pCon(J23100).
Promoter Activity Reporter (LacZ-alpha and RFP)
This part is the reverse of terminator B0013, a bacteriophage T7 terminator which was engineered to be bidirectional. Contains a poly A insertion upstream of a 20 bp stem-loop.
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.
Extracted from genomic DNA of ''S. cerevisiae'' using PCR.
ecol laccase from E. coli with T7 promoter, RBS and His-tag
E.coli laccase ORF with T7, RBS and HIS tag
Upstream construct of the Synthetics Multicellular Bacterium
trkD, a functional Kup (formerly TrkD) system took up Cs+ with a moderate rate and affinity
Escherichia coli cells which contain a functional Kup (formerly TrkD) system took up Cs' with a moderate rate and affinity. Kup is a separate K+ uptake system with relatively little discrimination in the transport of the cations K+, Rb+, and Cs'. Regardless of the presence or absence of Kup, K+-replete cells took up Cs'primarily by a very low affinity mode, proportional to the ratio of the Cs' and K+ concentrations in the medium. In our project, trkD is used to collect Cs-137 when recN is activated by radiation and start the transcription of trkD,in order to absorb this kind of radioactive source like Cs-137
This promoter is designed to bind to C2003.
Placement of the operator site was determined to optimize steric interference between C2003 and RNA polymerase.
This promoter was rationally designed and synthesized.
This part contains a constitutive promoter (J23106), a coding region of LacI protein with an LVA degradation tail, a terminator (B0015), a promoter repressed by LacI protein (R0011) a coding region of GvpA and GFP and a terminator (B0015) behind it. This part can be used to induce the strength of promoter (R0011) by IPTG and indicate the content of GvpA by fluorescence of GFP. In short, it can regulate the expression of GvpA. We can use different concentration of IPTG to regulate the strength of promoter (R0011) ahead GvpA Protein and GFP. And We can know the approximate concentration of GvpA by the fluorescence unit of GFP. To achieve this goal, we measured a curve to connect the concentration of IPTG and the fluorescence unit of GFP. Here is the curve:
We can use different concentration of IPTG to regulate the strength of promoter (R0011) ahead GvpA Protein and GFP. And We can know the approximate concentration of GvpA by the fluorescence unit of GFP. To achieve this goal, we measured a curve to connect the concentration of IPTG and the fluorescence unit of GFP.
Long matching input strand to the reporter formed by parts Part:BBa K779500 and Part:BBa K779502 or Part:BBa K779501 and Part:BBa K779502. Modifications: 5' IRD800 fluorophore, 3' phosphate. IDT DNA oligo order: /5IRD800/mCmA mCmCmC mAmCmU mCmCmU mCmUmC mCmCmA mCmCmA mAmCmU mAmUmC mCmA/3Phos/
3' phosphate added to protect from some 3' exonucleases (suggested by IDT). See more details at the MIT iGEM wiki: http://2012.igem.org/Team:MIT
PCR sil03F and sil03R on barnase region (348 bp, BglII/XhoI) Sub into pBca9145-Bca1089 (BglII/XhoI, 2063+696, L) Product is pBca9145-I716211 sil03F Forward BglII biobrick of barnase ccaaaAGATCTgcacaggttatcaacacgtttg sil03R Reverse XholI/BamHI biobrick of barnase gctagCTCGAGttaGGATCCttatctgatttttgtaaagg
xylR+PxylA: A xylose inducible promoter with its transcriptional regulator.
Yellow fluorescent protein (EYFP) reporter coding sequence without the Ribosome Binding Site. Modified with an LVA tail for rapid degradation of the protein and faster fall time for the emission. Annotated mutation cause a Q81L mutation that appears to not be near the active site.
BBa_E0032 yellow fluorescent protein is based on BioBrick part BBa_E0031. It has been modified to include a rapid degradation LVA tail, and includes the BioBrick standard assembly head and tail restriction sites. The RBS has been removed. The stop codon has been changed from TAA to a double stop codon TAATAA.
Modified from <bb_part>BBa_E0031</bb_part>
Kinetic characterization of 4-amino 4-deoxychorismate synthase from Escherichia coli. V K Viswanathan,; J M Green and; B P Nichols.
Strong promoter, strong RBS combination for high expression levels of proteins
Josh Atkinson, Mike Ferguson, and Ben Parker
-J23100+B0034 -Strong promoter, strong RBS combination for high expression levels of proteins Consensus constitutive promoter and RBS sequence-produces strongest possible expression.
It is a combination of strong promoter (J23100) and RBS (B0034).
PCaa3 promoter and native rbs from Synechocystis PCC6803
This is the 100bp sequence upstream the Caa3 gene start codon, it contains putative rbs and promoter regions. Caa3 codes for a putative cytochrome aa3 controlling protein and its mRNA levels have shown to oscillate in a circadian manner, peaking at hour 9.
the desition of taking just 100bp upstream C
this part was PCR amplified from Synechocystis chromosome, orf sll1898.
This part is used to screen the level of induction of the PF promoter (I746360). The PF promoter is induced by a class of phage activators (P2 ogr: I746350, PSP3pag: I746351, phiR73 delta: I746352).
The PF promoter was put upstream of I13504.
registry (I13504) and own construct (I746360)
Inducible araC-pBAD promoter regulating M256I CysE
The CysE M256I gene (K731010) is here regulated by the araC-pBAD promoter (K731200), which is inducible by arabinose.
K731200 and K731010 were inserted into pSB1C3 using a variation of the 3A Assembly, which added a PCR amplification step of the inserts before digestion to increase efficiency. The former was cut with E&S, the latter with X&P. pSB1C3 was digested with E&P.
crRNA-lock3-var1 (A new "lock" designed by our team)
crRNA(I714033) and taRNA(I714034) whose structure resemble that of J23078 (lock3) and J23066 (key3) but with a different matching regions to minimize the possible interaction with the key3-lock3.
autoinducer inactivation enzyme aiiA (no LVA)
[J23106,P0412]is a constitutive lacI repressor generator,Fluorescence can be observed with induced by IPTG. The RBS of E0240 is B0030,a weak GFP translational unit specialized to quantitively describe the behaviour of promotor in many cases. NOTE: R0011 don???t work well in high copy number plasmids.
Constitutive yeast promoter of medium strength compared to TEF1 promoter.
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">
Engineered FliC with insertable expression region
xanthosine methyltransferase CaXMT1-strep
xanthosine methyltransferase CaXMT1-strep from ''Coffea arabica'' followed by a strep-tag for purification and/or detection via western blot.
http://www.ncbi.nlm.nih.gov/nuccore/AB048793 synthesized by IDT, yeast codon optimized
Agrobacterium tumefaciens Bacteriophytochrome with RBS (E. coli Codon Optimised)
Agrobacterium tumefaciens bacteriophytochrome, codon optimised for expression in E. coli. In the presence of biliverdin the bacteriophytochrome will undergo a conformation change. This complex can then absorb red light(620-750 nm) to excite the complex and result in a phenotypic change from blue to green. This can be reversed by far-red light (700-800 nm) or will revert from green to blue over time. As E. coli does not produce biliverdin, heme oxygenase (Part BBa_K953000) can be coupled with this part to activate the oxidation of heme to produce biliverdin or by the addition of biliverdin to the cells.
cDNA of UV-B sensing protein UVR8 from Arabidopsis thaliana
UVR8 is responsible for UV-B detection in plants. In dark state UVR8 forms a dimer, which breaks into monomers upon exposure to UV-B light (280-315 nm). Later monomer induces transcriptional changes necessary for UV-B damage repairs and protection. In addition a BamHI site is inserted after ATG codon for fusions with other proteins (e.g. tetR-DNA binding domain), to use UVR8 as a UV-B sensing domain.
cDNA of UVR8 from Arabidopsis thaliana was provided by Roman Ulm, University of Geneva.
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
This is PBAD (BBa_I13453) mutant. -59 relative to the araC transcriptional start site was changed from C to T.
Constitutive promoter for LasR which can be repressed by TetR
Full-length lacZ with RBS and Terminator
RI7-odr10 (diacetyl) chimeric GPCR with promoter and terminator
BBa_K775003 encodes a chimeric diacetyl-sensing G-protein coupled receptor which can be functionally expressed in S. cerevisiae. This chimeric receptor is composed of the ligand-binding domain of the C. elegans 7-transmembrane receptor ODR-10, flanked by the N- and C- terminals of the rat OR RI7.
N-terminus: Olfactory receptor-like protein I7 (olr226), Rattus norvegicus (Rat) Ligand binding domain: C. elegans 7-transmembrane receptor ODR-10 U49449.1 C-terminus: Olfactory receptor-like protein I7 (olr226), Rattus norvegicus (Rat)
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
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.
4-coumarate - coenzym A ligase (4CL) + yeast consensus sequence
4-coumarate - coenzym A ligase (4CL) coding region from ''Arabidopsis thaliana'' preceeded by the yeast consensus sequence for improved expression.
Plasmid (template for PCR) provided by John A. Morgan, School of Chemical Engineering, Purdue University, West Lafayette, Indiana, USA http://www.ncbi.nlm.nih.gov/nucleotide/609339/ PMID: 15932991
BBa_K775002 encodes a chimeric isoamylacetate-sensing G-protein coupled receptor which can be functionally expressed in S. cerevisiae. This chimeric receptor is composed of the ligand-binding domain of the mouse OR1G1, flanked by the N- and C- terminals of the rat OR RI7.
N-terminus: Olfactory receptor-like protein I7 (olr226), Rattus norvegicus (Rat) Ligand binding domain: ORG1, (Homo sapiens) NM_003555 C-terminus: Olfactory receptor-like protein I7 (olr226), Rattus norvegicus (Rat)
Spot42 is a multitarget small RNA that mediates the discoordinate expression of the E.coli galactose operon and other sugar metabolic pathway,such as galK,nanC,ytfJ,srlA,which are the 5??? leader sequence of the corresponding metabolic enzymes. Take galK for example,Spot42 causes translation repression by base pairing to RBS in the 5??? leader sequence,then block the recognition of the antiSD sequence on 30S subunit. It???s a very strong pairing(up to 20bases),but it only causes 2.6 fold repression according to Johannes H. Urban and Jo?? rg Vogel,for it doesn???t result in the degradation of the RNA complex by RNaseE(ssRNA degradation) and RNaseIII(dsRNA degradation),both of them are major enzymes that causes RNA degradation in vivo.Spot42-galK complex degrades in a slow and presently unclear way. The base pairing is initiated by the recognition of seed region,which plays a significant role in the repression efficiency.
Digest pBca9145-I716206 (BamHI/AlwnI, 1694+553bp, Large) Digest pBca9145-Bca1114 (BglII/AlwnI, 1519+593bp, Small) Product is pBca9145-I716208.
This is the GLP-1 coding part along with the signal peptide sequence.
GFP fluorescence reporter to assay the activity of FimE K137007 and HbiF K880000 recombinases.
-IRR K137008 + inverted tetR promoter K137047 + IRL K137010 + GFP I13504 (MI275) -GFP fluorescence reporter to assay the activity of FimE K137007 and HbiF K880000 recombinases. Same components as K137058 with the inverted repeats reversed such that the external half sites are external to the flip region (IRR - ???flip region??? - IRL) GFP fluorescent reporter capable of assaying the functionality of fim regulatory recombinases; based on K137058. K137058???s invertible repeat sequences responsible for recombinase binding are ordered incorrectly; the regions are not identical, and have ???left??? and ???right??? components. K137008 (invertible repeat-right) and K137010 (invertible repeat-left) are mislabeled and belong in the order ???K137008-region to invert-K137010??? in accordance with the wild-type fim system. Repeat sequence in this part exist in the correct (wild-type) orientation.
LacI and RFP attached to a constitutive promoter for LuxI
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.
BglII and BamHI restriction sites with some flanking
http://openwetware.org/wiki/Silver:_Oligonucleotide_Inserts Oligonucleotide Inserts
This part contains a constitutive promoter (J23106), a coding region of TetR protein, a terminator (B0015), a promoter repressed by TetR protein (R0040) a coding region of GvpC and GFP and a terminator (B0015) behind it. This part can be used to induce the strength of promoter (R0040) by Tet and indicate the content of GvpC by fluorescence of GFP. In short, it can regulate the expression of GvpC. We can use different concentration of Tet to regulate the strength of promoter (R0040) ahead GvpC Protein and GFP. And We can know the approximate concentration of GvpC by the fluorescence unit of GFP. To achieve this goal, we measured a curve to connect the concentration of IPTG and the fluorescence unit of GFP. Here is the curve:
We can use different concentration of Tet to regulate the strength of promoter (R0040) ahead GvpC Protein and GFP. And We can know the approximate concentration of GvpC by the fluorescence unit of GFP. To achieve this goal, we measured a curve to connect the concentration of IPTG and the fluorescence unit of GFP.
FadA, gene for fatty acid degradation from Salmonella enterica LT2
FadA gene in S. enterica and in E.coli have similar function. Yet it is shown that S-FadA have far better efficiency compared to FadA in E.coli.(???S-??? is used to distinguish FadA gene in E.coli )
PCR sil04F and sil04R on BamHI methylase/endonuclease region. (657 bp, BglII/XhoI) Sub into pBca9145-Bca1089 (BglII/XhoI,2063+696, L) Product is pBca9145-I716210 sil04F Forward BglII biobrick of BamHI ccaaaAGATCTgaagtagaaaaagagtttattactga sil04R Reverse XholI/BamHI biobrick of BamHI gctagCTCGAGttaGGATCCctatttgttttcaactttatc
PCR sil04F and sil04R on BamHI methylase/endonuclease region. (657 bp, BglII/XhoI) Sub into pBca9145-Bca1089 (BglII/XhoI,2063+696, L) Product is pBca9145-I716210
Intermediate in two promoter screening plasmid
Adds an RBS to LacI protein coding region
3OC6HSL+LuxR dependent POPS/RIPS generator
produces pops and rips in the presence of luxR/3OC6HSL and the absence of lambda cI.
Constitutive promoter for LacI, repressed by TetR
This is composed of a promoter and a GFP reporter gene. This can produce green fluorescence and in theory the strength of the green flourence is much stronger when there is phosphorylated AgrA in the cell. This is used to test the basal activity of agr P2 promoter.
We used two subparts from the registry to produce the new part. BBa_I746104+BBa_E0840
fhlA: a transcriptional activator for enhanced hydrogen production
A to G (1906bp) (different from E. coli K-12 MG1655)
Full-length lacZ with an RBS ahead. This is a copy of the Part:BBa_I732017. Only here the part is cloned in the standard vector pSB1C3 instead of the pSB1A2.
We changed the backbone vector of the part from pSB1A2 to pSB1C3
This part contains an engineered theophylline riboswitch (RS) ([http://partsregistry.org/Part:BBa_K784005 BBa K784005]) and T7 RNA polymerase ([http://partsregistry.org/Part:BBa_I2032 BBa I2032]). The riboswitch contains a RBS and it will be exposed and starts translating the T7 RNAP only after inserting of theophylline. The RBS and the start codon of the gene are part of the riboswitch sequence and are responsible for its structure, so they are not to be changed. This part was constructed using fusion PCR, so the RS and the T7 RNAP are adjacent to one another without any restriction site between them. There is an additional stop codon at the end of the gene.
The riboswitch had to fused to the RNAP using assembly PCR.
Assembly PCR of [http://partsregistry.org/Part:BBa_K784005 BBa K784005] and [http://partsregistry.org/Part:BBa_I2032 BBa_I2032]. [http://partsregistry.org/Part:BBa_I2032 BBa I2032] was received from Ann K. Ganesan.
araC-Pbad regulated terephtalate periplasmatic binding proteine of the tripartite transporter family
bpul (laccase from Bacillus pumilus) with T7 promoter, RBS and HIS tag
bpul (Laccase from Bacillus pumilus) with T7, RBS and HIS tag
This is a coding part comprising the full open reading frame of Salmonella enterica LT2 vgrG gene (also known as STM0289 or tssI) linked via an haemagglutinin eptitope tag to a synthetic gene encoding an endolysin from the CD27 bacteriophage. The CD27 endolysin is believed to have high specificity against the cell wall of Clostridium difficile.
The vgrG section is derived from BBa_K895002 , which was used as a PCR template, and the endolysin section is derived from a synthetic gene designed by Neil Fairweather's group at Imperial College London.
Intermediate in screening plasmid v2 construction.
CinR (BBa_C0077) in complex with O3-C14:1-HSL (from CinI, BBa_C0076) binds to this promoter and activates transcription. This promoter contains the RBS: TGGAGG
The regulatory locus cinRI in Rhizobium leguminosarum conrols a network of quorum-sensing loci Lithgow, JK; Wilkinson, A; Hardman, A; Rodelas, B; Wisniewski-Dye, F; Williams, P; Downie, AJ MOL. MICROBIOLOGY 37(1): 81-97, 2000 Change log: the last 5 bases (gctaa) removed to allow for the correct spacing between the RBS (TGGAGG) and the start of the downstream gene after biobricks splicing.
GFP fused with AID-tag (ubuquitinase E3 OsTirI recoginition domain)
This part is a super folded GFP fused to an AID tag for its recignition by the ubiquitinase E3 that induces its degradation in the presence of auxin in the eukaryote cell. This part is a part of a device pattented by Kanemaki Masato, Kakimoto Tatsuo, Nishimura Kohei, Takisawa Haruhiko and Fukagawa Tatsuo for yeast and mammalian cells use (http://www.freepatentsonline.com/y2012/0115232.html). However the pattent does not cover the use for oviparian such as frogs and chicken. The GFP-AID contains 2 NLS domains that relocate the protein in the nucleus of the cells for a better signal concentration.
This part has been biobricked using PCR method and cloned in pSB1C3
This part have been extracted by PCR from the plasmid pNHK60 obtained from the riken registry.
Aditi Shrivastava, Jiwon Lee, Sarah Welch
putative terephtalate uptake operon2 AraProm_RBS_tctA-505_RBS_tctB-197_RBS_tphC
Spider Silk 1x Subunit "F" (Fewest tRNA codon optimized) with Met (ATG) added
Spider silk subunit optimized to use the fewest number of tRNA codons; contains two elasticity domains and one strength domain, also contains 5??? Met (ATG) codon.
Created using mutagenesis PCR to add upstream Met (ATG) to part BBa_K844003
4-coumarate--coenzyme A ligase (4CL) coding region
4-coumarate--coenzyme A ligase (4CL) coding region from ''Arabidopsis thaliana''.
This is I714031 with R0010 Promoter in the front.
tetracycline repressor from transposon Tn10 (+LVA)
Coding region for the TetR protein without the Ribosome Binding Site. Modified with an LVA tail for rapid degradation of the protein and faster fall time for the emission. TetR binds to the pTet regulator (BBa_R0040). aTc (anhydrotetracycline) binds to TetR and inhibits its operation.
References (unparsed) here: Elowitz, M. B. Transport, Assembly, and Dynamics in Systems of Interacting Proteins. Thesis, Princeton Univ., Princeton (1999). 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 Res. 1997 Mar 15;25(6):1203-10. PMID: 9092630 References (unparsed) here: Elowitz, M. B. Transport, Assembly, and Dynamics in Systems of Interacting Proteins. Thesis, Princeton Univ., Princeton (1999). 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 Res. 1997 Mar 15;25(6):1203-10. PMID: 9092630 BBa_C0040 TetR Protein is based on the TetR sequence from Elowitz's repressilator. It has been modified to include a rapid degradation LVA tail, and includes the BioBrick standard assembly head and tail restriction sites. The RBS has been removed. The stop codon has been changed from TAA to a double stop codon TAATAA.
This part contains the fluorescent protein mCherry attached to a degradation tag. It will decrease the amount of fluorescent protein over time.
We modified the AAV sequence to LVA with PCR.
eCFP + E. coli aggregation (Ag43) device induced by pBAD promoter.
In the presence of L-arabinose, pBAD promoter is induced and then wildtype Antigen 43 (Ag43) protein and eCFP (engineered cyan fluorescent protein)is produced. The wildtype Ag43 have 6 PstI sites. eCFP locates in the upstream of Ag43 for estimating the relationship between expressed protein amount and aggregation efficiency.
6 PstI sites exists in Ag43 coding site so we assembled this composite part without using PstI.
Ag43(BBA_K346007) was derived from E. coli K-12 strain by Peking 2010 iGEM.
yddV is a Diguanylate Cyclase-Genomic. The product of gene yddV has diguanylate cyclase (DGC) activity. DGC uses 2 GTP to form a Bis-(3???-5???)-cyclic dimeric guanosine monophosphate (c-di-GMP). C-di-GMP is a global second messenger in bacteria. Biofilm formation of E.coli is manipulable by varying c-di-GMP concentrations. When the c-di-GMP level stays low, there is little biofilm and the bacteria is dispersive. High concentrations of c-di-GMP promote bacteria to form more cellulose and fimbriae, which enhances the biofilm formation and decrease the motility of bacteria.
downstream construct of the synthetic multicellular bacterium
OriT-R (Origin of transfer for the R-plasmid nic region)
OriTR, the R plasmid nic region, is where the relaxosome nicks the plasmid and conjugative transfer by R plasmid machinery begins.
Inducible araC-pBAD promoter with GFPmut3b, strong RBS
This part has been used for testing and characterization of araC-pBAD promoter (part BBa_K731200). The part is composed of part BBa_K731200 and BBa_E0840
araC-pBAD from E. coli genome, GFPmut3b is from the registry (part BBa_E0840)
Isoamylacetate Chimeric GPCR with Fus1-EGFP reporter (2)
Contains both an early MfeI site and a late NsiI site. Cutting with Mfe and Spe and ligation to a BB part cut EX will form two mixed sites, neither of which can be recut. Cutting with Xba and Nsi and ligation to a BB part cut SP will again form two mixed sites, neither of which can be recut.
Should be grown in pSB1A2, as Nsi cuts both pSB2K3 and pSB1AK3 and Mfe cuts pSB4A3. 70 bp from Xba to Nsi or Mfe to Spe, the minimum for gel extraction. The middle is largely noise, to raise the size of the part.
This is a universal part designed to allow induction by HSL of whatever is assembled downstream of the part.
This part was constructed from registry biobricks.
Reporter top strand. Modifications: 5' RQ quencher, 3' Alexa florophore, all bases modified with 2'O Methyl (ordered as a DNA oligo with Us instead of Ts). IDT DNA oligo order: /5IAbRQ/mCmA mCmCmC mAmCmU mCmCmU mCmUmC mCmCmA mCmCmA /3AlexF488N/
Sequence designed to be orthogonal to the HEK293 transcriptome, and to have a high melting temperature. See more details at the MIT iGEM wiki: http://2012.igem.org/Team:MIT
pelB leader sequence attached to enzyme LC-Cutinase protein coding sequence in order to allow the cell to bring the enzyme to the periplasm for easier secretion.
Adding sequence of pelB in front of sequence for Cuntinase
http://aem.asm.org/content/78/5/1556.long Constructed from pelB leader sequence and protein coding sequence for LC-Cutinase
BBa_K775000 encodes a chimeric niacin-sensing G-protein coupled receptor which can be functionally expressed in S. cerevisiae. This chimeric receptor is composed of the ligand-binding domain of the mammalian GPR109A, flanked by the N- and C- terminals of the rat OR RI7.
The motA gene codes for a part of the flagellar motor complex. MotA proteins represent the proton conducting component of this molecular machine. Thus it is necessary for flagellar rotation.
*''motA'' was amplified from genomic DNA of ''E. coli'' DH10B via PCR using the following primers: **Fwd: GTTTCTTCGAATTCGCGGCCGCTTCTAGATGACCGGTGGAAGCCTTGG **Rev: GTTTCTTCCTGCAGCGGCCGCTACTAGTATCATGCTTCCTCGGTTGTCG
Vinay S. Mahajan, Brian Chow, Peter Carr
Generic spacer for ensuring a 70 bp distance between the end of the suffix of the BioBrick part containing the double terminator and the prefix of the BioBrick part containing the promoter of the new gene. Please, use the AlignX function of Vector NT to check for homology with the components in your plasmid before using this spacer.
The size of the spacer was choosed to meet the minimum length of a sequence that can be queried using the BLAST search engine. However, subsequences of it can be used to design shorter spacers. The sequence was selected from many more sequences randomly generated using the Random DNA Generator engine; the GC% parameter used as input was 50%. The sequences were selected based on the following constraints listed in their order of importance: the absence of any putative promoter regions, a low degree of homology with the Elowitz plasmid (whose components are widely used in our designs), no homology with other E.coli sequences as shown by BLASTN search results and the presence of a number of TAA stop codons. The second constraint was the most stringent leading to the elimination of most sequences. DE made the following changes to the original sequence in order to add stop codons in the -3 frame and more in the +2 frame (note, not all of these stop codons are UAA. Thus, if used in an organism that inserts an amino acid @ UGA or UAG the obvious will occur): T->A @ 85 T->A @ 42 C->T @ 79 A->T @ 64 A->T @ 31 T->A @ 34 C->A @ 37 Also, note that the above changes further reduce (the already very weak) homology to current NCBI-stored sequences. In the process of selecting the best sequence it appeared that a good alternative sequence for a spacer would be: AGGTTCTGATATGTAACTGTGCCCAATGTCGTTAGTGACGCATACCTCTTAAGAGGCCACTGTCCTAACA. The sequence contains no putative promoters and shows moderate homology with the 5' end of the Ampicillin resistance gene. However a strong promoter sequence starts 12 bp downstream of this sequence, and therefore the sequence presented above was preferred. The sequence is compatible (does not show significant homology) with the components in the Elowitz repressilator plasmid.
Randomly generated and optimized for several parameters (see Design notes).
c2 repressor from Salmonella phage P22 (+LVA)
The P22 c2 repressor protein coding sequence is a 720 base-pair sequence with the standard RBS-compatible BioBrick prefix and the standard BioBrick suffix sections on its ends. It binds to the P22 c2 regulatory sequence, BBa_R0053. The sequence contains a LVA tag for faster degredation.
References (unparsed) here: Vander Byl,C. and Kropinski,A.M. Sequence of the genome of Salmonella bacteriophage P22 J. Bacteriol. 182 (22), 6472-6481 (2000) References (unparsed) here: Vander Byl,C. and Kropinski,A.M. Sequence of the genome of Salmonella bacteriophage P22 J. Bacteriol. 182 (22), 6472-6481 (2000)
This is a coding part comprising the full open reading frame of the gene encoding the Salmonella enterica LT2 VgrG protein (also known as STM0279 or TssI). VgrG is an important component of bacterial Type VI Secretion Systems and is believed to locate at the very tip of the Type VI secretion device. Normally, therefore, this protein would be considered to be a secreted protein and to be located outside of the bacterial cell.
The Salmonella vgrG gene did not require any modification to meet RFC[10] standard.
Amplified by PCR from Salmonella LT2 genomic DNA.
The tRNA(Arg) is under the control of promoter tac. tRNA(Arg) expression is induced by 0.5mM IPTG when the OD600 of the culture reaches 0.3.
The correct processing to produce mature tRNA should be ensured.
LacI- Ptac derived from plasmid pTrc99B. tRNA(Arg) from E.coli ArgW operon.
downstream construct of the synthetic multicellular bacteria
shuttle vector pECPP11JS for recombination of genes of interest in yeast
the design considerations are coming soon
This part is made of the BioBricks XYZ by gibson assembly.
xylR+PxylA+RBS+ydcE+Ptms+RBS+ydcD: Cell Growth Inhibition Device
ent-Kaurene Oxidase from Gibberella fujikuroi
Genomic Sequence from Gibberella fujikuroi
pLasR is off by default and can be activated by LasR.
tphA2: Catalyzes together with tphA3 TPA to DCD
BcsB is involved in biosynthesis of cellulose, which is a significant constituent of the extracellular matrix observed during multicellular morphotype (rdar) growth. The BcsB protein that was indirectly inferred to bind c-di-GMP.
Escherichia coli str. K-12 substr. MG1655
tthl laccase from Thermus thermophilus with T7 promoter, RBS and His-tag
tthl (Laccase from Thermus thermophilus) with T7, RBS and HIS tag
Constitutive promoter for LuxI which can be repressed by LacI
T7 promoter-metG(truncated). This biobrick is constructed by putting the truncated metG (Met-RS) under the control of T7 promoter and lac operator. We have cloned metG from E.coli and the tRNA recognition domain of metG is truncated. Kana gene with start codon substituted for CGA is used to testify the function of mutated metG. When this biobrick and metY-CGA (BBa_K567016) are co-transformed into the cell, the cells can survive on the LBKana plate.
Inducible araC-pBAD promoter with GFPmut3b, weak RBS
This part has been used for testing and characterization of araC-pBAD promoter (part BBa_K731200) when using a weak RBS. The part is composed of part BBa_K731200 and BBa_E0240
Improved insertion site with two RNAse E cut sites
Shortened RNAse E site, insertion site, duplicate RNAse E site, and a trailing 5' hairpin (HP16 from Smolke et al., 2002).
Protein generator converting TIPS to the protein cI from lambda. Used as the input section for Quad Part Inverters Q01150 and Q01151.
3OC6HSL Sender Controlled by Lac Repressible Promoter
A lac repressible promoter driving production of an untagged LuxI enzyme which produces an AHL (3OC6HSL).
The BioBrick wtAID is an extended version of the existing AID BioBrick (BBa_K929001). It is built of 3 parts: CMV promoter (BBa_I712004), superAID and hGH polyadenylation signal sequence (BBa_K404108). AID: AID is known to be responsible for somatic hypermutation and the class-switch recombination of immunoglobulin in B cells. This enzyme of 28 kDa originally occurs in B cells but does also show activity after transfection into CHO cells. AID induces the deamination of cytidine to uridine at actively transcribed single strand DNA. The replacement of cytidine by uridine leads to a mismatch during DNA replication and integrates a single base substitution predominantly in the immunoglobulin genes. The AID motif is naturally terminated with the Nuclear Export Sequence (NES) that causes the protein to translocate from the nucleus to the cytoplasm. Additionally, upstream, dysfunctional Nuclear Localization Sequence (NLS) is located. Due to the fact that AID mutates the actively transcribed single stranded DNA, it is supposed that the direction of the enzyme to the inside of the nucleus would improve the mutation rate. That is why we called it superAID Functional NLS sequence: This part of the BioBrick directs the expressed protein into the nucleus, where it can mutate stronger. Kozak sequence Kozak consensus sequence is added upstream of the AID mutant to express the protein stronger. CMV promoter: CMV stands for cytomegalovirus. The CMV promoter is commonly used due to its very strong activity, and effectivity in a broad range of cell types. The BioBrick is therefore improved via addition of the strong promoter. hGH polyadenylation signal sequence: Polyadenylation is a significant part for the translation and stability of mRNA. In eukaryotes, it is part of the process that produces mature messenger RNA (mRNA) for translation. It, therefore, forms part of the larger process of gene expression. hGH terminator gives a signal to start polyadenylation in the translation process.
3 other Biobricks: CMV promoter (BBa_I712004), superAID (BBa_K929001) and hGH polyadenylation signal sequence (BBa_K404108).
Use this part to insert any 5?????TR of your gene of interest to screen for downregulation of your gene with small RNA. PCR amplify your 5???UTR with EcoRI and BamHI and clone into any backbone carrying this part. Digest whis part with EcoRI and BamHI and as RFP is replaced, this part functions as a red/white screening system. This allows you to create a reporter system with a fluorescent marker for screening for functional small RNAs. SYFP2 BBa_K864100 is situated downstrean of the RFP BBa_J04450 and is connected in frame with a 12 aa glycine-serine repeated linker BBa_J18922 , to prevent folding distruptions in the YFP or the 5???UTR. The first 6 nt of the glycine-serine linker is a BamHI restriction site (GGATCC), which translates into glycine-serine in E.Coli. When screening for working small RNAs, it's recommended to include the first 15 codons of the gene of interest.
Constructed from SYFP2 BBa_K864100 , linker BBa_J18922 and RFP BBa_J04450
Protein generator converting TIPS to the protein cII from p22. Used as the input section for Quad Part Inverters Q01530.
araC-Pbad - Arabinose inducible regulatory promoter/repressor unit
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.
Constitutive producer for TetR, repressed by LacI
This is PBAD (BBa_I13453) mutant. -67 relative to the araC transcriptional start site was changed from T to A.
pBADGLT:unstable gfp expression device activated by arabinose
Sifan Wang, Yizhen Yan, Jianzhao Chi, Qingshu Wu
This part contains Pbad promoter, RBS of 1.0 strength, GFP(lva) producer and double terminator. GFP(lva) will be produced after arabinose added. In the cell display part of our project, this part is used in part...part...part...
We use the unstable gfp for our project so the degree of fluorescence can be decreased after it get to a top point. In the cell display part, we can achieve the switch of diffrent numbers.
We built this part by using the biobricks from the dna distribution plates 2012.
Mycobacterium Phage Bxb1 gp35, DNA integrase
This part is the protein coding sequence which encodes DNA integrase of Mycobacterium phage Bxb1. The Bxb1 integrase is a DNA recombinase, more precisely a member of serine integrase family. It recognizes specific sequences, called attB and attP, and then integrate, invert, or excise depend on orientations of recognition sequences. We used this integrase to invert specific sequence contained in plasmid. This protein is well expressed in E.coli(strain MG1655). When it inverts DNA, the attB and attP sequences are changed into attL and attR, as common DNA recombinases do. Another protein called Bxb1 excisionase interacts with integrase and that complex flips back inverted DNA into original sequence regenerating attB and attP sequences.
Internal restriction endonuclease site is changed(two PstI sites, one XbaI sites) without changing amino acid sequence.
This part is derived by Mycobacterium phage Bxb1. And synthesized by Bioneer. http://www.ncbi.nlm.nih.gov/protein/NP_075302.1
TraR Coding Sequence from Agrobacterium tumefaciens
This part accepts POPS to generate EYFP under the medium strength RBS B0032.