The clp gene from Xanthomonas campestris
c-di-GMP Repressed DNA Binding Protein (Clp) Quorum sensing and bacterial cell signalling are mechanisms known to be utilised by bacteria to regulate their virulence. The RpfC-RpfG sensory system has been found to activate the production of virulence factors like extracellular enzymes and polysaccharides in Xanthomonas campestris. It has been also suggested that this system is involved in linking environmental stimuli, including DSF, with pathogenicity in Xanthomonas campestris. DSF is a cis-unsaturated fatty acid from the DSF family. The RpfC-RpfG two-component system is the most well understood one and is present in all Xanthomonads. http://www.ncbi.nlm.nih.gov/pubmed/?term=r+ryan+dsf+family http://www.ncbi.nlm.nih.gov/pubmed/?term=A+two-component+system+involving+an+HD-GYP+domain+protein+links+cell%E2%80%93cell+signalling+to+pathogenicity+gene+expression+in+Xanthomonas+campestris
The sequence had 1 PstI site that was mutated out by site-directed mutagenesis.
The source of this part is Xanthomonas campestris pv. campestris 8004 genomic DNA which was provided by the Robert Ryan lab in the Molecular Microbiology department of the University of Dundee, UK.
Plasmid in positive feedback verification: Pcons2-rbs-lacI-Ter-Plac-rbs-luxI-Ter-Pcons2-rbs-luxR-Ter-Prlux-rbs-rfp-Ter. It is a control plasmid for positive feedback circuit. It will produce rfp when there is enough IPTG.
Pcons2, lacI, Plac, luxI, Pcons2, luxR, Prlux, rfp, Ter
Gli1 bs + TEF2 + Untagged LexA activator on pRS306
This is a mutant T7 promoter that includes a lacO sequence. This is repressible by the LacI protein and is induced in the presence of IPTG. The promoter sits in the 5' region of the gene of interest and initiates transcription when the cell expresses T7 RNA polymerase. Be sure you are using a cell strain that includes this gene (DE3).
There are three portions to the T7 promoter: the recognition site, the melting box (or TATA box) and the initation site. Mutations in these sequences caused differential behavior from the wild type.
The part consists of a sequences that resemble a T7 promoter and the lacO sequence found in E. coli.
Olmecs: Inducible expression of 434 cI repressor
Plasmid1 in detector: Pcons2-rbs-luxR-Ter-Prlux-rbs-lacI-Ter-Plac-rbs-rfp-Ter It is a high level detection plasmid. It produces rfp when AHL is not so much.
Pcons2,luxR, Ter, Prlux, lacI, Ter, Plac, rfp, Ter
Fusion protein of SpyCatcher and RPD3 in an expression cassette
In this portion of the design, we aim to achieve population control by combining a cell-signaling system (luxR/luxI) and suicidal gene for controlling the number of E.coli. The population controlling function in the engineered E.coli can warn us of high-density E. coli congregations by emitting fluorescent proteins. Green fluorescent light means the Cry circuit is functional and the population density is under control, while red fluorescent gene signals E.coli overpopulation.
(I) Strand C (BBa_ K332021) 1. Strand C is synthesized from three fragments ??? (1) Ptet (2) RBS+LuxR (3) RBS+LuxI+double ter sites. 2. All of them are in the form of plasmids, so we digest them by appropriate restriction enzymes. 3. We first combine the last two fragments by ligation. The procedure is as follows: (1)Add the respective digestion products, vector plasmid, buffer, ligase to ddwater (2)React at 16??C for 30 minutes (3)Inactivate the ligase by incubation it at 80??C for 20 minutes (4)Store the products at 4??C 4. The products of ligation are then transformed into competent cells. The procedure is as follows: (1)2??l plasmids(ligation products) are added to 33??l competent cells (2)Shake gently for one second, and cool it on ice (3)Incubate it in 42??C water bath for 45 seconds (4)Spread the liquid to plate which corresponding antibiotics have been added to (5)37??C overnight 5. The next day we do a colony PCR for the transformed cells and have the PCR products electrophoresis to make sure that the two fragments have been combined correctly. 6. Now we separate the plasmids(RBS+LuxR+RBS+LuxI+double ter sites) from E. Coli, and digest it with restriction enzyme. 7. The first fragment (Ptet) is then combined with the above product by ligation, and transformed into competent cells by the same we have done early. 8. Because the length of Ptet to the remain segment is relatively too short, we cannot confirm whether they have combined correctly by just do an electrophoresis of the colony PCR products. If the electrophoresis result seems like that they have combined, we then send the product to sequencing.
1. LuxI protein (BBa_C0061) is the synthase that converts S-adenosylmethionine (SAM) into acyl-homoserine lactone (AHL). When the concentration of AHL reaches a threshold level, it binds to the N-terminal domain of a LuxR protein. Then the LuxR/AHL complex binds the Lux box within PtetR promoter (BBa_C0040), which then activates suicidal gene expression. 2. LuxR (BBa_C0062) is transcribed continuously by the constitutive promoter (BBa_J23106), and later works in conjunction with AHL. This design can control the concentration of LuxR protein in a steady phase, and the concentration of AHL/LuxR complex is only affected by the concentration of LuxI protein (BBa_C0061).
lexA3 is lexA (BBa_K864201) with a G85D mutation (G>A basepair change at position 254)
Produces LuxR protein which can sense 3OC6HSL in the media and activate transcription from R0062, the right hand Lux promoter
rhlR repressor/activator from P. aeruginosa PA3477 (no LVA)
Protein generator converting TIPS to the protein cI from 434. Used as the input section for Quad Part Inverter Q01520.
To edit. Pense ?? rajouter les linkers, je ne sais pas s'ils sont de igem ou s'il faut ajouter la s??quence ?? la registry. Check pour tous tes biobricks et dis-moi, please.
Zif268-HIV bs + LexA bs + mCYC + Zif268-HIV repressor (mCherryx2 tagged) on pRS306
Promoter sequence with recognition site for CueR transcriptional regulator protein.
The delta activator taken from phiR73 phage acts on a class of inducible promoters (parts I746360 to I746365), inducing their activity to varying degrees. The part sequence does already contain a ribosome binding site (B0034)!
The part does contain a RBS (B0034) already.
plasmid DNA supplied by Prof. Richard Calendar, University of California.
GFP - LasR anchor-inducer protein complex
This part contains a green fluorescent protein (GFP) as a non-reactive chemical compound (anchor) in order to neutralize the active reactive fixed to it, an inducer regulatory protein (LasR is responsible to induce pLas promoter in the presence of 6HSLO3 which is a chemical compound sythesized by an enzyme that LasI gene codes.) and a TEV protease cleavage site between them to assemble these two proteins. BBa_772001 and BBa_K772002 are assembled to be used as the trigger of detection in the whole system which ends with the intracellular transfection (or activation) of TEV protease. After the activation, it is planned that TEV protease will cleave this anchor-inducer protein complex to release free the reporter. In conclusion, in this system, the inducer would activate a transcription pathway on the targeted inducible gene or the reporter would indicate that there is a change in the whole system.
Cleavage site, LasR and GFP sequences are gathered from Registry.
Note that there is an RBS sequence just before the protein sequence in order to eliminate the need of addition of it.
This part was made by ligating K091145 to the 5' end of S03966.
These parts can be found in the registry.
Olmecs: Inducible expression of Lambda cI repressor
Fha is a RBS containing a binding site for the rsmA protein. Fixing this RBS, rsmA inhibits the transcription, which is recovered thanks to the rsmY iRNA, when it binds to the rsmA repressor.
This part was build following the standard construction protocol.
This RBS come from Pseudomonas aeruginosa.
RBS (BBa_B0030) placed in front of the ORF of XylR Transcriptional Regulator (BBa_I741005)
B0030 was used because of its stregnth, planning on expressing high levels of XylR
Composite - See BBa_B0030 and BBa_I741005
This part has two fused proteins downstream of an arabinose-inducible promoter: CpxR-IFP[1] and CpxR-IFP[2]. This construct was made in all orientations: - IFP[1]-CpxR + IFP[2]-CpxR (BBa_K1286009) - IFP[1]-CpxR + CpxR-IFP[2] (BBa_K1486010) - CpxR-IFP[1] + IFP[2]-CpxR (BBa_K1486011)
All orientations were made, so all dimerizations could be tested (see main page for biobrick numbers). Flexible linkers were used for each of the fused proteins, and an RBS was put before each one of them.
Prlux-rbs-cI-Ter. It produces cI when there is enough luxR and AHL.
Zif268-HIV bs + TEF2 + Untagged LexA activator on pRS306
This part has two fused proteins downstream of an arabinose-inducible promoter: CpxR-IFP[1] and CpxR-IFP[2]. This construct was made in all orientations: - CpxR-IFP[1] + CpxR-IFP[2] (BBa_K1286008) - IFP[1]-CpxR + CpxR-IFP[2] (BBa_K1486010) - CpxR-IFP[1] + IFP[2]-CpxR (BBa_K1486011)
Gli1 bs + MET25+ LexA repressor (CFPx2 tagged) on pRS304*
send P-AI-1 signaling molecules out, using Mnt(or cI) protein repress the pMnt/lac( or plux/cI) hybrid promoter
PgolTS-golS-PgolB-tetR-Ter-PtetO-rfp-Ter. If there exist gold ions, rfp will not be produced.
PgolTS-golS-PgolB, tetR, PtetO, rfp, Ter
The Pseydomonas quinolone signal (PQS) as well as its precursor 2-heptyl-4-quinolone (HHQ)are very important in mechanisms controlling virulence in Pseudomonas aeruginosa. P. aeruginosa has a key role in Cystic Fibrosis (CF). PQS can initiate signalling for various processes like virulence, motility, and biofilm formation. A good number of these processes are facilitated by PqsR, which is a transcriptional factor. An activated PqsR will go to the nucleus of the cells and activate the respective genes. http://www.ncbi.nlm.nih.gov/pubmed/21539583 http://www.ncbi.nlm.nih.gov/pubmed/?term=pqsr-dependent+and+pqsr-independent+regulation+of+motility+and+biofilm+formation+by+pqs http://www.ncbi.nlm.nih.gov/pubmed/12393198
The sequence did not have any restriction sites to remove so it was immediately PCRed out of the genome and had the registry prefix and suffix added to it. It was then ligated into the pSB1C3 vector.
The source of this part is the genome of Pseudomonas aeruginosa PAO1 provided by the Robert Ryan lab in the Molecular Microbiology department of the University of Dundee, UK.
Plasmid2 in detector: Prlux-rbs-cI-Ter-PcI-rbs-lacI-Ter It is a low level detection plasmid. It produces rfp when AHL is enough.
Zif268-HIV synthetic activator (untagged)
This is a synthetic transcriptional activator for use in yeast. It contains the VP16 activation domain, the Zif268-HIV DNA-binding domain, the SV40 nuclear localization sequence, and the ADH1 terminator. It binds to the Zif268-HIV binding sites, activating transcription downstream.
From David Drubin in the Silver Lab at Harvard.
Plasmid in positive feedback verification: Pcons2-rbs-lacI-Ter-Plac-rbs-luxI-Ter-Pcons2-rbs-luxR-Ter-Prlux-rbs-rfp-rbs-luxI-rbs-luxR-Ter It is a positive feedback plasmid using luxI and luxR as key to feedback. Introducing IPTG will produce rfp, luxI and luxR, and luxI together with luxR will work back to enhance production of rfp.
Pcons2, lacI, Plac, luxI, luxR, Prlux, rfp, Ter
Protein generator converting TIPS to the protein cI from lambda. Used as the input section for Quad Part Inverters Q01150 and Q01151.
LacI repressor (temperature-sensitive mut 265) (+LVA)
Mutated from BBa_C0012 Repressor, LacI (LVA+) to be temperature sensitive. Mutation from Chao, et al., 2002: Gly265->Asp (GG(803)T->GAT)
This expression casette constitutively produces the LuxR regulatory protein that can respond to the quorum sensing molecule 3OC6. It also produces a LacI repressor with a mutation at position 12 that results in an isoleucine. The mutant repressor has been shown to have an increased affinity for its operator.
Fusion protein of TALE3 and a SpyTag in an expression cassette
rbs+appY (encoding a DNA-binding transcriptional activator)
This is the coding sequence of appY from Escherichia coli JM109 preceded by a synthetic ribosome binding site (rbs). It encodes a transcriptional regulator related to anaerobic energy metabolism. Overexpression of the gene has been reported to result in enhanced lycopene production by E. coli (Kang et al., 2005).
Aditi Shrivastava, Jiwon Lee, Sarah Welch
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)
pGAL1 + Untagged LexA activator on pRS305
Quorum sensing is the mechanism of communication between bacteria via synthesizing and detecting chemical compounds in the media. With these mediators, the promoters and their yield can be tuned or regulated. This part represents an alternative quorum sensing sender device which is designed to induce PoPS production in the parts including pLas such as BBa_K772004. LasI is an enzyme generator gene sequences which synthesizes 6HSLO3 (AHL) in order to induce a PoPS response in the pLas promoter. pLas promoter works in the presence of both 6HSLO3 and LasR protein.
Note that there is an RBS sequence just before LasI; in order to be easily used with quorum sensing receiver devices such as BBa_F2620 or BBa_K772004.
LasI sequence is gathered from Registry.
Protein generator converting TIPS to the protein cI (HK022). Used as the input section for Quad Part Inverter Q01500
This part is a combination of the two parts K091155 and K091144.
These two parts contain parts that are in the registry and DNA built from oligos.
T7 RNA polymerase with RBS (ribosome-binding site) and NLS (nuclear localisation signal). NLS is used to target the protein to the cell nucleus.
DNA was isolated from BL21 and PCR was performed to attach RBS and NLS.
Tet repressor generator (version1, TERM-)
This part is similar to [http://partsregistry.org/wiki/index.php/Part:BBa_I732082 BBa_I732082], but there is a mutation induced by PCR. We are convinced that the mutation has nothing to do, and this part works as well as [http://partsregistry.org/wiki/index.php/Part:BBa_I732082 BBa_I732082].
pTEF2 + Untagged LexA activator on pRS305
pMET25 + Untagged LexA activator on pRS305
Olmecs: Inducible expression of HK022 cI repressor
Rbs-cI(scarless) It is an improvement of cI(BBa_C0051) by adding a rbs.
It is an improvement of cI(BBa_C0051) by adding a rbs.
we construct a CI mutant which is derivated from CI857(ind-)
make a CI mutant which is not degenerated by RecA*
Bas Stringer (Sequence by Paul van Dieken)
This is a synthetic transcriptional activator for use in yeast. It contains the VP16 activation domain, the LexA DNA-binding domain, the SV40 nuclear localization sequence, and the ADH1 terminator. It binds to the Lex operator, activating transcription downstream.
From David Drubin in Silver Lab at Harvard.
Zif23-GCN4 engineered repressor (+LVA, C2000 codon-optimized for E.coli)
Variant of Zif23-GCN4 (see BBa_C2000). CDS has been optimized (via VNTI) for expression in enteric bacteria. ssrA tag (LVA) added.
This protein should form a dimer and bind a specific operator site, thus repressing transcription.
Protein Engineering Gods from the Pabo Lab
lsr promoter of LuxS/AI-2 signaling pathway in Salmonalla
Quorum sensing is a process of bacterial cell-to-cell communication involving the production and detection of extracellular signaling molecules called autoinducers. And autoinducer-2 (AI-2) has been proposed to serve as a 'universal signal' for interspecies communication. In the LuxS/AI-2 signaling system of Salmonella Typhimurium, AI-2 response involves ATP binding cassette transporter encoded by genes named Lsr (LuxS regulated). And plsr is member of lsr operon which could be repressed by LsrR. In our project, we set this promoter part in front of a blue pigment as a reporter monitoring the presence of AI-2.
The lsrD gene was isolated from the genome of Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 using PCR. We added restriction enzyme sites when designing primers, and the assembly is done through the use of restriction sites (cutting and ligating).
We extracted the genomic DNA of Salmonella enterica subsp. enterica serovar Typhimurium str. LT2, and used PCR to isolate the promoter plsr.
Prlux-rbs-rfp-rbs-luxI-rbs-luxR-Ter. It produces rfp, luxI and luxR when there is enough luxR and AHL.
This part has a fused protein downstream of an arabinose-inducible promoter: IFP[2]-CpxR. Biobrick BBa_K1486014 has IFP[1]-CpxR with an arabinose promoter. This construct was made in both orientations: BBa_K1486013 is CpxR-IFP[2] (with an arabinose promoter).
Both orientations were made, as well as for IFP[1] so all dimerizations could be tested (see main page for biobrick numbers). Flexible linkers were used to fuse both proteins, and an RBS was put before each one of them.
tet-controlled LacI generator with constitutive TetR expression cassette
This BioBrick contains a tetracyclin-inducable LacI generator, which is followed by a constitutive TetR expression cassette. Since E. coli does not produce TetR, the expression cassette has to be present on the plasmid; otherwise the tet-controlled promotor will be constitutively active.
We decided to put the constitutive expression cassette behind the inducible LacI generator to minimze readthrough from the constitutive expression.
Assembled from parts K142046 and I739001 by standard cloning techniques.
The ogr activator taken from P2 phage acts on a class of inducible promoters (parts I746360 to I746365), inducing their activity to varying degrees. The part sequence does already contain a ribosome binding site (B0034)!
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr
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.
Lov-HTH (light sensor with Ptet repression) light->PtetO repressed
This page is about a light sensor and regulator protein: Lov-HTH. Lov-HTH is a artificial light sensor designed by iGEM12 Fudan Lux team. This protein include two function domain, Lov the light sensing domain and HTH the DNA binding domain.
The HTH motif swap is the main issue in our design. We carefully choose the HTH sequence and swap it into the original amino acid sequence.
The sequence is reverse translated from the protein EL222 http://www.rcsb.org/pdb/explore/explore.do?structureId=3P7N which is from Erythrobacter litoralis. The DNA is synthesized by Invitrogen company.
This is a TetR protein generator regulated by the constitutive promoter J23111. The protein has a LVA tag for degradation.
Zif268-HIV bs + LexA bs + mCYC + Zif268-HIV repressor (mCherryx2 tagged) on pRS304*
This part is the twin of the non-compatible biobrick BBa_K1486008 rendered compatible by mutating the two illegal restriction sites. However, all experiments were run on the aforementioned biobrick.
Site-directed mutagenesis was used to remove the restriction sites in the sequence.
http://www.addgene.org/browse/article/8177/ which was then mutated by us.
Coding sequence for LacI modified with a different rate of translation than LacI, RBS
LacIm from Basu et al."A synthetic multicelluar system for programmed pattern formation", Nature. 2005 Apr 28;434(7037):1130-4
rhlR repressor/activator from P. aeruginosa PA3477 (+LVA)
Debra Lin, Srini Devadas, David Gray, Ronny Krashinsky, and Chris Zheng Liu, Boopers
Transcriptional regulator, in complex with N-butyryl-HSL, RhlR binds to the Rhl promoter
(1/28/04 Edit Jamboree) (0) No BB sites (1) ATG (2) TAATAA (3) Blast checks out (4) No codon changes (5) Maybe chassis compatability (check by experiment) (6) Codon usage not optimal (but looks OK) (7) ssrA tag added
Yichong wang, Ruichen, Xihao liao and Junjie zhang
BBa_K1072002 is the generator fo NADH dehydrogenase which is an enzyme following chemical reaction: NADH + H+ acceptor \rightleftharpoons NAD+ + reduced acceptor
Mag is a RBS containing a binding site for the rsmA protein. Fixing this RBS, rsmA inhibits the transcription, which is recovered thanks to the rsmY iRNA, when it binds to the rsmA repressor.
Pcons2-rbs-lacI-Ter. It constantly produces lacI.
Olmecs: Inducible expression of P22 c2 repressor
Fusion protein of TALE2 and a SpyTag in an expression cassette
Coming soon, if I am allowed to edit this.
It keeps saying I have no permission to edit this.
Protein generator converting TIPS to the protein LacI. Used as the input section for Quad Part Inverters Q01121 and Q01122.
Lactose inducible lysis cassette expression
Constitutive LacI expression ensures tight repression of lysis.
Lysis cassette is cloned from Lambda Zap II genomic DNA.
Strong RBS + LacI repressor + double terminator
This part contains a strong ribosome binding site, the LacI repressor and a double terminator (BBa_B0030 + BBa_C0012 + BBa_B0014). This brick comes from the BBa_K292005. The LacI repressor represses pLac promoter from the E.coli lactose operon. So LacI repressor and this bio-brick are really reliable to use when we want to control a gene expression by using pLac promoter. In fact when LacI is not expressed, pLac is activated, and when there is an expression of LacI, pLac is inhibited. We can easily design a system to induce the transcription of LacI by using another operon as the tetracycline operon and creates a failed feedback mechanism for pLac inhibition and so, for gene expression inhibition. The LacI (BBa_K292006) already contains a strong RBS and a double terminator, so this brick is ready to use! History: In E.Coli, pLac is a part of the lactose operon. LacI is constantly expressed and represses the pLac. In the presence of Lactose, LacI is inhibited and pLac is active and allows the transcription of beta-galactosidase. Lactose can be substituted by IPTG (Isopropyl β-D-1-thiogalactopyranoside).
Part:BBa_B0030, Designed by Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003. Group: NCBS, Prather Lab Part:BBa_C0012, Designed by Grace Kenney, Daniel Shen, Neelaksh Varshney, Samantha Sutton Group: Registry Part:BBa_B0014, Designed by Reshma Shetty Group: Registry (2003-07-16)
Gli1 bs + LexA bs + mCYC + LexA repressor (mCherryx2 tagged) on pRS304*
This part is composed of an arabinose-inducible promoter for superfolded GFP attached by a linker to CpxR (on the C terminus of CpxR).
We had to test both attachments, N and C terminus of the CpxR.
The arabinose promoter and the sfGFP were obtained from the registry, while the CpxR was obtained from the E coli genome.
The pag activator taken from PSP3 phage acts on a class of inducible promoters (parts I746360 to I746365), inducing their activity to varying degrees. The part sequence does already contain a ribosome binding site (B0034)!
Coding region for the HK022 bacteriophage cI protein. cI binds to the HK022 pR regulator (BBa_R0050). It represses transcription of the protein encoded by the sequence 3' to the pR region. This coding sequence does not contain a RBS.
References (unparsed) here: Carlson NG, Little JW. Highly cooperative DNA binding by the coliphage HK022 repressor. J Mol Biol. 1993 Apr 20;230(4):1108-30. PMID: 8487297. Mao C, Little JW. Mutations affecting cooperative DNA binding of phage HK022 CI repressor. J Mol Biol. 1998 May 29;279(1):31-48. PMID: 9636698. References (unparsed) here: Carlson NG, Little JW. Highly cooperative DNA binding by the coliphage HK022 repressor. J Mol Biol. 1993 Apr 20;230(4):1108-30. PMID: 8487297. Mao C, Little JW. Mutations affecting cooperative DNA binding of phage HK022 CI repressor. J Mol Biol. 1998 May 29;279(1):31-48. PMID: 9636698. Derived from STHK022N . Response from John Little (Arizona) regarding the start of HK022 cI From: <jlittle@email.arizona.edu> Date: Tue Jan 21, 2003 4:39:21 PM US/Eastern To: "Drew Endy" <endy@MIT.EDU> Subject: RE: hk022 cI start (naive question)? Hello Drew and Michael, I seriously doubt that the extra 27 aa are part of CI. It doesn't make sense in terms of the biology, for sure. In any case, the protein we characterized starts where Carlson and Little stated; as I recall, oR3 partially overlaps the start of cI. I have a vague memory of some possibly interesting biology, having to do with multicopy plasmids. I don't recall the findings themselves. Anyway, one possible explanation was that this extra N-terminal addition was made (e.g. from the pRE promoter, or from a message that arose from transcription around the entire plasmid), making a protein with altered functions. We never followed it up. Good luck John -- Original Message -- Date: Sun, 19 Jan 2003 15:26:26 -0500 Subject: hk022 cI start (naive question)? Cc: elowitm@rockefeller.edu To: jlittle@u.arizona.edu From: Drew Endy <endy@MIT.EDU> Hi John, I'm sitting here with Michael Elowitz and we're working through the sequence for HK022 cI. We noticed that the annotation from NC_002166 includes an "extra" 81 base pairs upstream of what we thought of as the actual start. It looks like this extra DNA extends into the PR regulatory region. We're wondering what's going on here? I'm guessing this is well documented somewhere. Thanks for any pointers/info! Drew It is unknown whether there is cross-talk between this repressor and Lambda cI regulatory region, 434 cI regulatory region and P22 regulatory region.
lasR activator from P. aeruginosa PAO1(+LVA)
coding region for lasR protein, which accepts chemical signal AI-1 (made by lasI protein)
Mutated 480 from g to a to remove PstI site
Genbank: NC_002516 (www.ncbi.nlm.nih.gov)
Fusion protein of TALE1 and a SpyTag in an expression cassette
complete tet./ dox. inducible promoter from Invitrogen vector pcDNA/FRT/TO, T-Rex system
IPTG-on tetracycline-off pulse generator with LacI mutant (T276F) and TetR expression cassette
==IPTG-on/tetracycline-off switch circuit== ===Short description:=== This part contains a tetracycline-controlled LacI IS expression cassette followed by a constitutive TetR expression cassette. The LacI IS mutant is almost identical to the LacI transcriptional regulator except for the difference that it is not able to bind IPTG or allolactose due to a mutation; it therefore can not be activated by induction with these substances. Since it recognizes the same motif in the lac promotor region, it strongly represses transcription of all genes regulated by promotors with lacI binding site even if IPTG or allolactose are present. It is used to terminate the expression of proteins under lac control if IPTG can not be removed from the cell rapidly. ===Detailed description:=== ====LacI IS mutants are uninducible repressors of lac-controlled promotors==== Expression of the lac operon in E. coli is tightly controlled by LacI, a protein, which binds to a repressor binding site within the promotor and disables transcription by obscuring the promotor region. When bound to DNA, LacI is in the tetrameric form, which consists of two dimers interacting at the end distal from the DNA binding site. Upon binding of allolactose or IPTG, the tetramer breaks down into two dimers and the affinity for the repressor binding site is greatly reduced; the lacI IPTG complex will diffuse away from the repressor binding site, leaving the promotor accessible. As a result of decades of genetic and structural studies, the function of lacI is now understood on the molecular level (1, 2). Mutational experiments have identified residues, which abolish IPTG response upon mutation (3). Furthermore, the x-ray crystal structure of LacI with bound IPTG has allowed the identification of residues that interact with IPTG and which are promising targets for mutagenesis (1). [[image:jr_pulsegen_1.jpg|frame|none| A LacI repressor tetramer, residues R197 and T276 are shown in red. B IPTG bound to the inducer binding site of the lac repressor, residues R197 and T276 are shown in green. Molecular graphics was generated from coordinate set [http://www.rcsb.org/pdb/explore.do?structureId=1LBH 1lbh] (1) using [http://www.cgl.ucsf.edu/chimera/ UCSF Chimera].]] We decided to mutate residues R197 and T276, which are located in the IPTG binding groove, contact IPTG and have been shown to produce the lacI IS mutation in previous genetic experiments. We decided to generate a set of eight mutated lacIs, in which we replaced either R197 with alanine or phenylalanine or T276 with alanine or phenylalanine or both in all possible combinations. lacIIS-1: R197A lacIIS-2: R197F lacIIS-3: T276A lacIIS-4: T276F lacIIS-5: R197A T276A lacIIS-6: R197A T276F lacIIS-7: R197F T276A lacIIS-8: R197F T276F All mutants appeared to be equally efficient in preliminary genetic experiments with full repression even at elevated concentrations of 10mM IPTG. Please see the chapter on the switch circuit in the [http://2008.igem.org/Team:ETH_Zurich/Wetlab/Switch_Circuit ETH 2008 Wiki] for details. ====Purpose of this BioBrick==== This BioBrick allows pulsing of protein expression under lac-controlled promotors. It is especially useful if change of medium to remove the inducer is not an option (such as inside a fermenter/chemostat). Since the signal for stop of expression is independent from the induction of expression, pulses of different duration can be tested without any modification of the pulse generator construct. ===References:=== (1) Lewis, M., Chang, G., Horton, N. C., Kercher, M. A., Pace, H. C., Schumacher, M. A., Brennan, R. G., and Lu, P. (1996) Crystal structure of the lactose operon repressor and its complexes with DNA and inducer. Science 271, 1247-54. (2) Friedman, A. M., Fischmann, T. O., and Steitz, T. A. (1995) Crystal structure of lac repressor core tetramer and its implications for DNA looping. Science 268, 1721-7. (3) Suckow, J., Markiewicz, P., Kleina, L. G., Miller, J., Kisters-Woike, B., and Muller-Hill, B. (1996) Genetic studies of the Lac repressor. XV: 4000 single amino acid substitutions and analysis of the resulting phenotypes on the basis of the protein structure. J Mol Biol 261, 509-23.
During site-directed mutagenesis, the codons to be mutated were replaced with the most highly utilized codons in E. coli to prevent complications from the use of rare codons. The lacI IS sequences were analyzed for BioBrick restriction sites within the coding sequence to ensure their compatibility.
The pulse generator precursor has been assembled from tetracycline-controlled lacI generator [http://partsregistry.org/wiki/index.php?title=Part:BBa_K142046 K142046] and the constitutive TetR expression cassette [http://partsregistry.org/Part:BBa_I739001 I739001] using standard cloning techniques. The lacI IS mutant presented derives from site-directed mutagenesis, which was performed on the precursor [http://partsregistry.org/wiki/index.php?title=Part:BBa_K142047 K142047] by PCR, subsequent DpnI digest and transformation. The following primers were used for mutagenesis: R197F forward CGGCGCGTCTGTTTCTGGCTGGCTG R197A forward CGGCGCGTCTGGCGCTGGCTGGCTG R197F reverse CAGCCAGCCAGAAACAGACGCGCCG R197A reverse CAGCCAGCCAGCGCCAGACGCGCCG T276F forward GGATACGACGATTTTGAAGACAGCTC T276A forward GGATACGACGATGCGGAAGACAGCTC T276F reverse GAGCTGTCTTCAAAATCGTCGTATCC T276A reverse GAGCTGTCTTCCGCATCGTCGTATCC
Gli1 bs + LexA bs + mCYC + Zif268-HIV repressor (mCherryx2 tagged) on pRS303
A similar part is available in registry. we designed this as we faced some problems in CFP expression with the earlier part. we have tested this part and it is functional.
pGAL1 + Untagged Zif268-HIV activator on pRS305
lac-controlled GFP-LVA generator with constitutive lacI expression cassette
We put the LacI generator behind the lac-controlled expression cassette to minimze readthrough.
Subcloned from BioBricks using standard molecular biology techniques.
Plac-rbs-tetR-Ter. It produces tetR when there is enough IPTG or less lacI.
Pcons2-rbs-lacI-Ter-Plac-rbs-luxI-Ter It produces luxI when there is enough IPTG.
This is a TetR protein generator regulated by the constitutive promoter J23109. The protein has a LVA tag for degradation.
marR(G95S) carries a G>A mutation at position 284 which gives a amino acid change G95S. Has been mutated from BBa_K864200.
tetracycline repressor from transposon Tn10 (+LVA)
June Rhee, Connie Tao, Ty Thomson, Louis Waldman.
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.
Elowitz, M. B. Transport, Assembly, and Dynamics in Systems of Interacting Proteins. Thesis, Princeton Univ., Princeton (1999)
PphoA promoter is a phosphate-responsive promoter. In high concentration of phosphate, it will be repressed while in low phosphate concentration, it will be activated.
Vibrio harveyi repressor protein LuxO and phosphorelay protein LuxU genes. LuxO and LuxU are regulators of luminescence in Vibrio harveyi.
LuxO and LuxU are found within the operon, therefore we have decided to keep these genes together. The natural RBS was also kept.
GenBank Accession# L26221 LuxO Protein ID# AAD12736.1 LuxU Protein ID# AAD12737.2 Jeremy A. Freeman and Bonnie L. Bassler. Sequence and function of LuxU: a two-component phosphorelay protein that regulates quorum sensing in Vibrio harveyi. 1999. Journal of Bacteriology. Feb 1999, p. 899-906.
AHL regulated PoPS generator: Las receiver device
Quorum sensing is the mechanism of communication between bacteria via synthesizing and detecting chemical compounds in the media. With these mediators, the promoters and their yield can be tuned or regulated. This part represents an alternative quorum sensing receiver device, which is able to generate a PoPS response in the presence of 6HSLO3 produced by LasI, instead of BBa_F2620. This will allow iGEMers to establish communications between different bacteria colonies and it makes simple to generate easy regulated complex systems. This part is assembled with Las promoter system to improve the current quorum sensing devices and parts which commonly base on Lux promoter system.
Note that pLas has no RBS afterwards, the assembling methods must be selected after this consideration.
pLas and LasR sequences are gathered from Registry.
Plasmid in positive feedback verification: Pcons2-rbs-lacI-Ter-Plac-rbs-luxI-Ter-Pcons2-rbs-luxR-Ter-Prlux-rbs-rfp-rbs-luxR-Ter. It is a positive feedback plasmid using luxR as key to feedback. Introducing IPTG will produce rfp and luxR, and luxR will work back to enhance production of rfp.
Protein generator converting TIPS to the protein TetR. Used as the input section for Quad Part Inverter Q01140.
mnt repressor (weak) from Salmonella phage P22 (+LVA)
dimeric repressor from enterobacteriophage p22 with dissociation constant of 3*10^-8. sequence was translated with the E.Coli K12 codon usage table, and nucleotides 95-97 was changed from AAT to AAC to avoid the cut site GAATTC (94-99). the mutation R10->K10 (ctg->aaa) decreases the binding affinity of the mnt protein to its operator by approximately 12,000 fold (Kd: 2.5e-12 to 3e-8)
RsmA is a coding sequence from Pseudomonas aeruginosa which increases the pathogenicity by allowing type VI secretion. Taken alone, it does not code for a dangerous protein and it does not increase the risk level.
Tetracycline Repressor with Consitutive Promoter BBa_J23103
A transcriptional regulator TetR (BBa_C0040) is driven by constitutive promoter family members (BBa-J23103, BBa-J23109, BBa-J23116, and BBa-J23106), which can be used to tune the expression level of constitutively expressed TetR.
The SsrA at the end of BBa_C0040 is not present in our design.
The source of this part are all from the registry distributions.
mnt repressor (strong) from Salmonella phage P22 (+LVA)
wild type dimeric repressor from enterobacteriophage p22 with dissociation constant of 2.5*10^-12. sequence was translated with the E.Coli K12 codon usage table, and nucleotides 95-97 was changed from AAT to AAC to avoid the cut site GAATTC (94-99). Identification of Functionally Important Residues in the DNA Binding Region of the MNT Repressor Kendall, KL; Sauer, RT The Journal of Biological Chemistry, 264(23):13706-13710, 1989
lac-controlled CFP generator with constitutive lacI expression cassette
hybrid lambda PR-lac promoter, repressed by lambda cI and lacI, plus lacZ
This part is a hybrid promoter designed to be repressed by either lambda cI or LacI. It consists of the the PRM-PR promoter region of bacteriophage lambda, including cI binding sites OR1, OR2 and OR3 as well as the -35 and -10 sites of both PRM and PR, fused via an introduced XhoI site to the LacI binding site of the lac promoter. (In this version, the lacZ' gene encoding the N-terminal 76 amino acid residues of LacZ has also been included to facilitate testing.) This approximately preserves the relationship between the -10 promoter region and and the LacI binding site. Our hope is that this promoter will be effectively repressed in the presence of either lambda cI or LacI. However, this has not been properly tested at the time of writing. We can say that good LacZ activity is observed in the presence of IPTG, indicating that the basic promoter function of lambda PR appears to be intact. Our sequencing also suggests two single nucleotide mutations within OR2 as compared to published sequence, which may be natural variants or PCR-induced mutations; in the latter case, they may or may not have some effect on cI binding. Note also that this construct includes the PRM promoter, which ought to be functional in the reverse direction.
This part includes the entire lambda PRM-PR region, with cI binding sites OR1, OR2 and OR3 and the -35 and -10 sequences of both promoters (PRM and PR). An XhoI site was introduced by PCR, and the DNA was joined to the region of the lac promoter beginning with the LacI binding site. The distance between the LacI binding site and the PR -10 site in the fusion is approximately the same as that between the LacI binding site and its native -10 site; thus we hope that LacI will be able to repress PR in our construct. However, this has yet to be properly tested.
The lambda-derived portion (bases 4 to 78) is derived from lambda DNA by PCR using primers based on published sequence (Genbank accession J02459, gi:215104). The lac portion (bases 84 to 353) is derived from E. coli BL21 genomic DNA using primers based on published sequence (Genbank accession J01636, gi:146575).
Gli1 bs + TEF2 + LexA repressor (CFPx2 tagged) on pRS304*
lac-controlled RFP generator with constitutive lacI expression cassette
Rbs-tetR(scarless) It is an improvement of tetR(BBa_C0040) by adding a rbs.
It is an improvement of tetR(BBa_C0040) by adding a rbs.
pBAD to tetR coding region, lacI coding region, and mCherry reporter
Links the pBAD promoter, which is induced by arabinose and inhibited by araC, to a tetR coding region with an RBS, a LacI coding region with an RBS, and an mCherry reporter. Activation of this pathway results in tetR production, lacI production, and mCherry production.
Strength of RBS may affect the strength of output.
This part has a fused protein downstream of an arabinose-inducible promoter: CpxR-IFP[2]. Biobrick BBa_K1486012 has CpxR-IFP[1] with an arabinose promoter. This construct was made in both orientations: BBa_K1486015 is IFP[2]-CpxR (with an arabinose promoter).
This photoreceptor was assembled by Strickland et al., and consists of a LOV (light-oxygen-voltage) domain of Avena sativa phototropin1 (AsLOV2) that senses blue light, fused to the trpR- DNA binding domain of the transcription factor trp repressor. The resulting protein is named LovTAP: LOV- and tryptophan-activated protein. LOV domains bind a flavin-mononucleotide (FMN) or flavin-adenine-dinucleotide (FAD) cofactor, which are used in a wide variety of metabolic pathways as cofactors in redox reactions and are available in most organisms. The cofactor has a broad absorption spectrum, with a maximum at 450 nm. Besides, the core of the LOV domain is often flanked by amino- or carboxy-terminal helices, termed A???α and Jα, respectively In the LovTAP construction, AsLOV2 domain via its carboxyl-terminal Jα ???helix was ligated to an amino-terminal truncation of TrpR. The resulting protein has a domain-domain overlap with a shared helix. Thus, photoexcitation would change the conformation of the protein, in turn changing the stability of the shared-helix-domain contacts. Under the presence of light, absorption of a photon leads to the formation of a covalent adduct between the flavin mononucleotide (FMN) cofactor and a conserved cysteine residue in the AsLOV2 domain, which results in conformational rearrangements in LovTAP. This change impacts the affinity of the shared helix for the two domains: disrupting the contacts between the shared helix and the LOV domain and enabling the association of the shared helix with the TrpR domain, which establishes DNA-binding affinity and LovTAP can then bind DNA as an homodimer, repressing the transcription of the genes downstream of its binding sites. In the dark, when the shared helix contacts the LOV domain, the TrpR domain's DNA-binding affinity decreases and LovTAP is in an inactive conformation.
We decided to synthesize a new LovTAP part, that in comparison with the Part:BBa_K191003 that is already at the registry, has the following differences: 1. The 2 PstI restriction sites were removed from the coding region of LovTap. 2. We included a punctual mutation to change the ILE427 by a PHE427, as was proposed by the model results of the team iGEM09_EPF-Lausanne. With this mutation LovTAP should react faster and the conformational change should be more stable (the protein stays in the active form for longer, under light induction). 3. The part does not include a promoter. We eliminated the inverting regulator sensitive to LacI and CAP protein(Part:BBa_R0010). According to the report of EPF-Laussane team, the expression levels of LovTAP under the inverting regulator, doesn??t seem to show differences to the induction with IPTG. Thus, we decided to remove it and as we know that the level expression of LovTAP must be low (because at high levels of expresion, there is unspecific regulation under darkness and light states), we plan to test weak promoters to choose the best option and include it in the construction. 4. The RBS was changed from a strong (Part: BBa_B0030) to a medium strength (Part:BBa_B0032), thus expecting lower levels of lovTAP protein. 5. The stop codon tga was changed for two taa.
Chiameric protein assembled by Strickland et al. Reference: Strickland, D., Moffat, K., & Sosnick, T. (2008). Light-activated DNA binding in a designed allosteric protein. Proceedings of the National Academy of Sciences, 105(31), 10709. National Acad Sciences. Retrieved from http://www.pnas.org/content/105/31/10709.full.
Prlux-rbs-rfp-rbs-luxR-Ter. It produces rfp and luxR when there is enough rfp and luxR.
THe pTet promoter can be repressed by TetR repressor protein. The cI protein generated can repress plambda(R-O12)(K094002) promoter. This part lacks a terminator
The pTet promoter and the rbs comes from the registry, The cI protein comes from lambda phage
The part accepts POPS to produce TetR. This sequence encodes the ribosome binding site and tetracycline repressor protein from the plasmid pASKIBA3.
(correct) temperature sensitive promoter
RBS(B0032)+T3 DNA-directed RNA polymerase
This part is the coding region of MerR, which is a mercury responsive regulator from Tn21.
We cloned this part from plasmid NR1, which was a gift from Anne O. Summers.
Full Gli1 Repressor (tagged with mCherryx2)
Kozak+Sin3p RD+Gli1 DBD+mCherryx2+SV40NLS+tADH1
This part is composed of an arabinose-inducible promoter for superfolded GFP attached by a linker to CpxR (on the N terminus of CpxR).
This is the ''virG'' response regulator from the virA/G receptor system. The virA/G receptor system is used by ''Agrobacterium tumefaciens'' to detect phenolic substances secreted by plants. In presence of these substances, ''virG'' is activated by the ''virA'' receptor and induces the transcription of genes under the control of a ''vir'' promotor. Normally, ''virG'' needs the ''rpoA'' RNA polymerase subunit from ''A. tumefaciens'' to work in ''Escherichia coli'' but this ''virG'' BioBrick is mutated, so works with the ''rpoA'' subunit from ''E. coli''.
synthesized by Mr. Gene, so optimated codon usage, removal of illegal restriction sites
TP901 cI repressor from phage TP901-1 (+LVA)
Dimeric repressor from bacteriophage TP901-1 with dissociation constant of 2*10^-9. Sequence was translated with the E.Coli K12 codon usage table, and nucleotides 84-86 was changed from TTC to TTT to avoid the cut site GAATTC (81-86). Bronsted, L., Hammer, K., Johansen, A.H., 2003. Identification of operator sites of the CI repressor of the phage TP901-1: evolutionary link to the other phages. Virology 311, 144-156. cI sequence slightly different from part C0051. NCBI locus: NC_001416
Removed LVA tag from BBa_C0052 via PCR. In pSB1AK3 currently.
Transcription Factor Binding Site from Cyanobacterial Pcpc560
Prlux-rbs-lacI-Ter. It worked out an expression of lacI in the presence of luxR and AHL.
PgolTS-golS-PgolB-tetR-Ter If there exist gold ions, tetR will be produced.
Arsenic Promoter (ArsR regulated) with own RBS
This part is an inverting regulator sensitive to LacI and CAP. It contains two protein binding sites. The first binds the CAP protein, which is generally present in E.coli and is asocciated with cell health and availability of glucose. The second binds LacI protein. This part also produces LacI protein as an oscillator which results that as LacI protein is synthesized, the promoter wil be inhibited until the presence of IPTG. The inhibition will decrease the production of LacI which leads to the synthesis of this inhibitor. >In the absence of LacI protein and CAP protein, this part promotes transcription. >In the presence of LacI protein and CAP protein, this part inhibits transcription. >LacI can be inhibited by IPTG. >LacI is coded by BBa_C0010
Note that this part is not working unless IPTG is present in the media.
This is a MoClo converted version of BBa_C0079
This is a MoClo converted version of BBa_C0079, which is the coding region for the lasR protein, which accepts chemical signal AI-1 (made by lasI protein). It has two MoClo fusion sites flanking it.
We had to design the proper MoClo fusion sites to flank this part.
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)
MC7-rbs-tetR-Ter-PtetO-rbs-rfp-Ter-Plac-rbs-tetR-Ter-Pcons2-rbs-lacI-Ter It is a test of our suicide circuit for microcystin.
MC17, tetR, PtetO, rfp, Plac, Pcons2, lacI, Ter
lsrR coding gene with ribosome binding site B0034.
Zif268-HIV bs + ADH1 + Gli1 repressor (CFPx2 tagged) on pRS304*
This part is adapted from K283006 with a terminator added to the end of the device.
The CI, terminator, pLambda promoter and the chez coding region are self designed The rbs, and the pLux promoter are from the registry
lasR activator from P. aeruginosa PAO1(no LVA)
A composite part with coding sequence for LacI-LVA together with ribosome binding site (RBS) and a double transcriptional terminator. Fore use in regulation of LacI-repressed genetic elements, by combining the part with an appropriate promoter.
This part is a reconstruction of BBa_K292006. BBa_K292006 has the same theoretical sequence, but the DNA available through the registry does not match this sequence. We thus offer this part as a replacement.
Pcons2-rbs-luxR-Ter-Prlux-rbs-lacI-Ter. It produces lacI when there is enough AHL.
This is a two-protein generator (cI and lasR) without transcriptional terminator. Strong RBS (efficiency=0.6).
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.
pGAL1 + Null synthetic activator on pRS305
Note that this is a ligation that we made onto a pRS305 vector, which does not conform to Biobrick standard. As such, there is some remnant of the Biobrick ends but
This part contains a lambda promoter that is constitutively on and can be repressed by CI protein. cheZ protein is responsible for the dephosphorylation of cheY protein in bacteria flagella movement. This part lacks a terminator.
the terminator is not included in this part
From non-biobrick plasmid, flanked by biobrick prefix and surfix.
cinR activator from Rhizobium leguminosarum (+LVA)
In complex with O3-C14:1-HSL (made by BBa_C0076), CinR binds to the Cin promoter (BBa_R0077) and activates transcription.
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: original STOP: tag -> taATAA
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.
represillator of Elowitz and Leibler (2000)
P_tet->NheI->RBS->luxI->double terminator
This part generates AHL (Source organism: V. fischeri). The promoter is P_tet which induced by Tetracycline or by its analog anhydrotetracycline (aTc). Before the promoter there is the prefix site and after the promoter there is a restriction site of NheI, so the promoter can be replaced by a restriction enzyme reaction. In addition, after the NheI restriction site, there is an RBS and luxI which is coding to AHL. At the end of the part, there is double terminator and the Suffix site.
The part has been sequenced and has been checked with a detector strain which detects AHL and produce color. We know for sure that it produces AHL.
P_tet: BBa_I759002- we used only the promoter, without the rest of the part (spacer (including the RBS)-cro complex) and added few bp as a spacer. RBS: BBa_B0030. luxI: BBa_C0061 but we removed the LVA tag and added stop codon: "tag". Double terminator BBa_B0015
The promoter PAtpI has its origin in Bacillus subtilis. It is responsible for the expression of atpA gene (ATP synthesis) during the first 30 min of germination [https://www.ncbi.nlm.nih.gov/pubmed/25661487 Sinai''et al.'']. This gene is part of an operon, therefore the promoter region in front of the first protein coding gene (atpI) in this operon was chosen. The region was checked for the binding of sigma factors and transcription factors with DBTBS. No binding factors were found with the highest significance level. In our project we wanted to find a constitutive ON promoter for our ciprofloxacin resistance casette. In the following part we put the promoter PAtpI in the pSB1C3 backbone to make it available to other iGEM teams.
Ordered as gBlock. IDT (Integrated DNA technologies).
Gli1 bs + ADH1+ LexA repressor (CFPx2 tagged) on pRS304*
Tetracycline Repressor with Consitutive Promoter BBa_J23109
The SsrA site at the end of BBa_C0040 is not present in our design.
The source of this part is based on the registry distribution
BBa_K1072023 is a induced promotor, originating from S.cerevisiae.
PgolTS-golS-PgolB-tetR-Ter-PtetO-rfp-Ter-Plac-tetR-Ter It is part of our test of suicide circuit by gold ions.
PgolTS-golS-PgolB, tetR, PtetO, rfp, Plac, Ter
ssrB protein under pTet promoter. The ssrB protein can activate the prosseA promoter. The pTet promoter can be repressed by TetR repressor protein. The ssrB coding region does not contain any terminator
The ssrB coding region is self-designed The rbs and the pTet promoter are from the registry
Promoter sequence with recognition site for ArsR transcriptional regulator protein. ArsR binds to the promoter sequence in the absence of As and releases on binding of As, therby activating transcription.
This part has a fused protein downstream of an arabinose-inducible promoter: CpxR-IFP[1]. Biobrick BBa_K1486013 has CpxR-IFP[2] with an arabinose promoter. This construct was made in both orientations: BBa_K1486014 is IFP[1]-CpxR (with an arabinose promoter).
Both orientations were made, as well as for IFP[2] so all dimerizations could be tested (see main page for biobrick numbers). Flexible linkers were used to fuse both proteins, and an RBS was put before each one of them.
Zif268-HIV bs + LexA bs + mCYC + Gli1 repressor (mCherryx2 tagged) on pRS303
The ferric uptake repressor of Bacillus subtilis called YqkL is a regulator that when in contact with iron will bind and repress a regulatory sequence (the Fur box).
We sequenced the Fur box. This can be added in front of genes to provide regulation when in contact with iron.
Promoter sequence with recognition site for ZntR transcriptional regulator protein.
LasR coding region and a double terminator
The biobricks BBa_C0079 and BBa_B0015 where used for this biobrick.
toxicity-gene activator from Vibrio cholerae
shortened BBa_J07008. Typical ToxR-periplasm-region subsitutions are done @ aa 211. explanation of ToxR domains @ http://model.mit.edu/igem/images/d/d7/Kolmar95.pdf ELIMINATE XbaI SITE PLZZZZZ
Tetracycline Repressor with Consitutive Promoter BBa_J23106
The source of this part is based on the parts in the registry distribution
cI protein generator without terminator (strong RBS, efficiency=0.6)
cI: Vinay S Mahajan, Brian Chow, Peter Carr, Voichita Marinescu and Alexander D. Wissner-Gross RBS: Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
The marR gene encodes a repressor of the marRAB operon
Amplified from MG1655 (Accession NC_000913) chromosome using primers listed below marR_BB_F TTCGAATTCGCGGCCGCTTCTAGATGAAAAGTACCAGCGATCT marR_BB_R GGCCGCTACTAGTATTATTACGGCAGGACTTTCTT
GFP - LasR protein complex with flag tags
This part contains a green fluorescent protein (GFP) as a non-reactive chemical compound (anchor) in order to neutralize the active reactive fixed to it, an inducer regulatory protein (LasR is responsible to induce pLas promoter in the presence of 6HSLO3 which is a chemical compound sythesized by an enzyme that LasI gene codes.) and a TEV protease cleavage site between them to assemble these two proteins. BBa_772001 and BBa_K772002 are assembled to be used as the trigger of detection in the whole system which ends with the intracellular transfection (or activation) of TEV protease. After the activation, it is planned that TEV protease will cleave this anchor-inducer protein complex to release free the reporter. In conclusion, in this system, the inducer would activate a transcription pathway on the targeted inducible gene or the reporter would indicate that there is a change in the whole system. This part contains flag tags extra from BBa_K772006 in the GFP region; because it is planned to be used in the Co-IP/SDS-PAGE assay to be isolated.
Pcons2-rbs-luxR-Ter. It achieves a constant expression of luxR, the AHL combining protein Pcons2 is BBa_J23100
Rbs-lacI(scarless) It is an improvement of lacI by adding a rbs.
It is an improvement of lacI by adding a rbs.
Gli1 bs + LexA bs + mCYC + LexA repressor (mCherryx2 tagged) on pRS306
P_ompC->NheI->RBS->luxI->double terminator
This part generates AHL (Source organism: V. fischeri). The promoter is P_ompC which induced by the Taz receptor which is cloned on a different plasmid as a different patr. Before the promoter there is the prefix site and after the promoter there is a restriction site of NheI, so the promoter can be replaced by a restriction enzyme reaction. In addition, after the NheI restriction site, there is an RBS and luxI which is coding to AHL. At the end of the part, there is double terminator and the Suffix site.
P_ompC: BBa_R0082 RBS: BBa_B0030 luxI: BBa_C0061 but we removed the LVA tag (gctgcaaacgacgaaaactacgctttagtagcttaataactctgatagtgctagtgtagatctc) and added stop codon: "tag". Double terminator BBa_B0015
Amplified from MG1655 (accession NC_000913) chromosome using primers listed below (annealing sequences are underlined) lexA_BB_F TTCGAATTCGCGGCCGCTTCTAGATGAAAGCGTTAACGGCCAG lexA_BB_R CGGCCGCTACTAGTATTATTACAGCCAGTCGCCGTT
This part has a fused protein downstream of an arabinose-inducible promoter: IFP[1]-CpxR. Biobrick BBa_K1486015 has IFP[2]-CpxR with an arabinose promoter. This construct was made in both orientations: BBa_K1486012 is CpxR-IFP[1] (with an arabinose promoter).
Both orientations were made, as well as for IFP[2] so all dimerizations could be tested (see main page for biobrick numbers). Flexible linkers were used to fuse both proteins, and an RBS was put between the promoter and the cds.
Pseudomonas putida, phiR73 phage, E.coli
penI repressor from Bacillus licheniformis (+LVA)
Since penI is being ported from Bacillus Licheniformis, the sequence was changed within the coding region to reflect codon usage in E.Coli K12. Codons were mapped to the codon of the same usage rank in E.Coli.
LacI repressor (temperature-sensitive mut 241) (+LVA)
Mutated from BBa_C0012 Repressor, LacI (LVA+) to be temperature sensitive. Mutation from Yabuta, 1995: Ala241->Thr (G(730)CG->ACG)
This part contains a LacI repressor and a terminator (BBa_C0012 + BBa_B0014). The LacI repressor repressed pLac promoter from the E.coli lactose operon. So LacI repressor is really reliable to use when we want to control a gene expression by using pLac promoter. In fact when LacI is not expressed, pLac is activated, and when there is an expression of LacI, pLac is inhibited. We can easily design a system to induce the transcription of LacI and creates a failed feedback mechanism for pLac inhibition. History: In E.Coli, pLac is a part of the lactose operon. LacI is constantly expressed and represses the pLac. In the presence of Lactose, LacI is inhibited and pLac is active and allows the transcription of beta-galactosidase. Lactose can be substituted by IPTG (Isopropyl β-D-1-thiogalactopyranoside).
When we design the bio-brick it was impossible to obtain a functional bio-brick by using the plasmid SB1AK3. But it's worked with a pSB1A2.
Part:BBa_C0012, Designed by Grace Kenney, Daniel Shen, Neelaksh Varshney, Samantha Sutton Group: Registry Part:BBa_B0014, Designed by Reshma Shetty Group: Registry (2003-07-16).
Zif268-HIV bs + TEF2 + Gli1 repressor (CFPx2 tagged) on pRS304*
RsmY codes for a iRNA which binds and inhibits the rsmA repressor.
Zif268-HIV bs + MET25+ Gli1 repressor (CFPx2 tagged) on pRS304*
Elowitz RBS. CinR CDS (no LVA tag) and terminator. Same as I0468 but no LVA tag
Tetracycline Repressor with Consitutive Promoter BBa_J23116
The tetR-HNS2-90 protein fuses the polymerization demain of histone-like nucleoid-structuring (H-NS) protein to tetR. Thus, in E.coli, when TetR-HNS2-90 binds the TetR-specific DNA motif, it will recruit native H-NS to bind to adjacent DNA, which may lead to local gene silencing. This fusion protein is driven by constitutive promoter family members (BBa-J23103, BBa-J23109, BBa-J23116, and BBa-J23106). Experimental data indicates that TetR-HNS2-90 is more effective than TetR on transcription repression.
RsmA is a transcription repressor protein who fixes fha or mag RBS. The transcription is recovered by the rsmY iRNA which binds the rsmA protein.
Lov-tetR (light sensor with Ptet repression) light->PtetO repressed
This part code a protein as a light sensor and repressor. With the existence of this protein, expression downstream the PtetO promoter will be repressed under the 450nm blue light. This protein, which is actually a well designed fusion protein, include two domain. The Lov domain is from Avena sativa phototrophin1(AsLOV2). The tetR is from the tetracycline resistance, which could combine the tetracycline and enable the expression of the tetA. We carefully designed the linker between these two parts, and fulfilled our sensor part in our biowave circuit.
This part is designed for our biowave project.
This coding sequence is form the Avena sativa phototrophin1(AsLOV2) and tetracycline resistance. The whole gene is synthesized by Invitrogen company.
Kozak+Sin3p RD+LexA DBD+CFPx2+SV40NLS+tADH1
Protein generator converting TIPS to the protein cII from p22. Used as the input section for Quad Part Inverters Q01530.
MC31-rbs-tetR-Ter-PtetO-rbs-rfp-Ter-Plac-rbs-tetR-Ter-Pcons2-rbs-lacI-Ter It is a test of our suicide circuit for microcystin.
MC31, tetR, PtetO, rfp, Plac, Pcons2, lacI, Ter
araC arabinose operon regulatory protein (repressor/activator) from E. coli (+LVA)
coding region for the araC gene, used to make araC protein for use in positive or negative regulation of TIPs output (see also R0080, R0081)
GenBank: NC_002655 (www.ncbi.nlm.nih.gov)
PcI-rbs-lacI-Ter. It produces enough lacI when there is less cI.
HasI is the sigma factor under the has operon system
The HasI gene is originally found in the Serratia marcescens. We obtained the gene sequence from the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/nuccore/X81195.2) and synthesised the gene
This is a TetR protein generator regulated by the constitutive promoter J23106. The protein has a LVA tag for degradation.