LuxPQ from Vibrio harveyi LuxP is a ABC-type sugar transport system periplasmic protein (Protein ID = YP_001447483.1). LuxQ is a histidine kinase (Protein ID = YP_001447484.1).
LuxP and LuxQ are found in the operon, thus we decided to keep them together. We kept natural RBS in front of LuxP, as well as the intrinsic terminator. Silent mutations were performed on a EcoRI site located within LuxP, EcorI site within LuxQ, and XbaI site within LuxQ.
GenBank Accession: NC_009784 Vibrio harveyi ATCC BAA-1116 chromosome II, REGION: 579654..580751. Neiditch MB, Federle MJ, Pompeani AJ, Kelly RC, Swem DL, Jeffrey PD, Bassler BL, Hughson FM. Ligand-induced asymmetry in histidine sensor kinase complex regulates quorum sensing. Cell. 2006 Sep 22;126(6):1095-108. Neiditch MB, Federle MJ, Pompeani AJ, Kelly RC, Swem DL, Jeffrey PD, Bassler BL, Hughson FM.
Lux based Sender/Receiver/Reporter system (LVA-)
Analogue of the V. Fischeri quorum-sensing system. Built using Biobrick components. Includes LuxR and LuxI under the control of their natural promoters and includes a YFP output device on the same transcript as LuxI (Same as I13264 but LuxI has no LVA tag)
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
Pcons2-rbs-lacI-Ter-Plac-rbs-luxI-Ter It produces luxI when there is enough IPTG.
luxR protein generator under Plambda regulation
This device produces luxR constitutively under the strong promoter Plambda, in absence of cI repressor. Plambda can be turned off by cI repressor and luxR production cannot continue. Strong RBS (efficiency=0.6). Artificial 39 bp terminator (efficiency=0.99).
Tlanslational unit of Colicin Lysis Protein (for colicin-E3)
Colicin lysis protein allows colicins to be released. The mechanism how the colicin lysis protein allows colicin release has not been fully elucidated, but it is sure that this protein raise membrane permiability and cause quasilysis.[1] Colicins are a cytotoxins which are released to environment and kill other related strains.
Colicin-E3 cleave 16SrRNA at a specific site. This activity is inhibited by immunity protein for Colicin-E3(BBa_). When lysis protein for Colicin-E3(BBa_) is co-expressed, Colicin-E3 is emited to environment and kill other strain. Colicin is a type of bacteriocin produced by Escherichia coli.
This gene sequence includes an EpCAM (Epithelial p? Cell Adhesion Molecule) antigen binding domain, OmpA transmembrane protein (dock) and TEV protease N terminus. It is planned to use this subpart with BBa_772, which contains only TEV protease C terminus differently instead of N terminus, in order to induce a signal reaction in the case of EpCAM presence. EpCAM is an natural antigen which is found high significantly in the cases of some breast and colorectal cancer subtypes. On the upstream region of protein sequence, there are a constitutive pLac promoter and appropiate RBS. To see how this part works: Experience.
There are two protein sequences between domains and the dock: One between the binding domain (antibody) and dock and one between the part of TEV protease.
CD225 (EpCAM antibody) is commonly used for the test of presence of EpCAM in the laboratories. The sequences of OmpA and TEV protease terminuses are acquired from the Registry.
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 device accepts PoPS as input and produces the LuxI enzyme. This enzyme produces 3OC6HSL.
the cheY* gene is installed in front of mRFP operon. cheY is the gene coding for cheY protein which controls flagella movement. This sequence, however, is mutated to give a mutant cheY protein which will not respond to the presence of cheZ protein. This cheY* gene is without a promoter, rbs or terminator.
the cheY* gene is without an rbs nor a terminator
Generates lasI in response to aTc input.
pGAL1 + Flag + Brho + odr-10 + GFP + ADH1Te + pFUS1 + BFP + ADH1Te
GFP and aiiA are repressed Lux promoter GFP and aiiA induced by AHL. GFP will express if the amount of AHL exeed the capability of aiiA.
This device senses the C4HSL concentration in the media and produces output PoPS. RhlR production is controlled by constitutive P(BIa). This part is the C4HSL analog of F2621.
rhlR repressor/activator from P. aeruginosa PA3477 (no LVA)
Outputs varying levels of aiiA as part of HSL/aiiA measurements. aiiA is untagged. Similar to I13204
Prlux-rbs-rfp-rbs-luxI-rbs-luxR-Ter. It produces rfp, luxI and luxR when there is enough luxR and AHL.
This gene sequence includes both of EpCAM Binding Docks; BBa_K772001 and BBa_K772002. Thus, this composite part is the main component of CTC (Circulating Tumor Cell) detection system. Docks include an EpCAM (Epithelial Cell Adhesion Molecule) antigen binding domain, OmpA transmembrane protein (dock) and TEV protease C or N terminus. Hopefully, after binding to the EpCAM antigen, TEV protease domains will fix together to form the full structure in order to activate the signalling pathway. (For more information: BBa_K772003) EpCAM is an natural antigen which is found high significantly in the cases of some breast and colorectal cancer subtypes. On the upstream region of protein sequences, there are a constitutive pLac promoter and appropriate RBS. To see how this part works: Experience.
There are two protein sequences between domains and the dock: One between the binding domain (antibody) and dock and one between the domain of TEV protease.
This is a two-protein generator (cI and lasR) without transcriptional terminator. Strong RBS (efficiency=0.6).
Pcons2-rbs-luxR-Ter. It achieves a constant expression of luxR, the AHL combining protein Pcons2 is BBa_J23100
Complete CDS of Rat olfactory protein mRNA, clone I7.
MC31-rbs-luxI-Ter If there are microcystin, the luxI will be produced.
Lux Receiver (I13263 with reversed part order)
Receiver circuit similar to I13263 but with the order of the reporter and luxR generator reversed
3OC6HSL Sender Controlled by Lac Repressible Promoter
A lac repressible promoter driving production of an untagged LuxI enzyme which produces an AHL (3OC6HSL).
High Population->mRFP (pLac->LuxI; pLacIQ->luxR; pLux->mRFP;)
Quorum sensing circuit under a pLac promoter. Population turns red (mRFP) once quorum sensing kicks in.
Double terminator not used after LuxI because it is unnecessary and has the potential to make the plasmid more unstable.
Built through assembly of BBa_K084014 and BBa_J10000.
Rbs-luxI(scarless) It is an improvement of luxI(BBa_C0061) by adding a rbs.
It is an improvement of luxI(BBa_C0061) by adding a rbs.
Plux-rbs-CI-terminator-plambda P(O-R12)-rbs-cheZ
This is a part in which the expression of cheZ is repressed by increasing AHL concentration in the environment in the presence of luxR protein. In other circumstances the cheZ gene is constitutively expressed. The cheZ gene lacks a terminator.
luxR protein generator under Plambda regulation (TERM-)
luxR protein generator without transcriptional terminator. This generator is under Plambda repressible promoter. Strong RBS (efficiency=0.6).
Anti-fluorescein scFv (one mutation, S101A) with transcriptional terminator.
aiiA (LVA-) protein generator driven by ptet
Produces aiiA protein from the tet promoter. The aiiA is untagged and should be capable of degrading any intracellular AHL.
autoinducer inactivation enzyme, AaiiA (+RBS)
This protein generator produces the enzyme LasI. This enzyme produces 3OC12HSL. This is the 3OC12HSL analog to part F1611.
HasR is the membrane-bound receptor in the Has system, a heme acquisition system originally from the Serratia marcescens. The HasR protein is involved in binding with a heme-loaded HasA hemophore to trigger downstream HasS and HasI. It has been shown that the N terminal of HasR is especially crucial for signal transduction. With the heme-loaded hasA bound to hasR, it induces a conformational change in HasR that causes the activation of HasS, an anti-sigma factor. HasS then releases HasI (sigma factor) that was initially bound to it and HasI ultimately is able to trigger expression of genes under control of pHas (part number).
The HasS gene is originally found in the Serratia marcescens. We obtained the gene sequence from the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/nuccore/X81195.2) and synthesised the gene.
This is a cheZ gene that can be induced by luxR protein under high AHL concentration. cheZ protein is responsible for the dephosphorylation of cheY protein in bacteria flagella movement.
composited from E. Coli cheZ gene and lux promoter
Colicin-E3, its immunity protein, and colisin lysis protein is constitutively expressed.(under BBa_J23110) When this device works, Colicin-E3 is released to media, and kill E. coli strains which does not have immunity protein.
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.
The HasR gene is originally found in the Serratia marcescens. We obtained the gene sequence from the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/nuccore/X81195.2) and synthesised the gene.
Produces LasR protein which can sense AI-1 in the media and activate transcription from R0078, the right hand Lux promoter
lasR activator from P. aeruginosa PAO1(no LVA)
Colicin-E3, its immunity protein, and colicin lysis protein is constitutively expressed strong.(under BBa_?J23101) When this device works, Colicin-E3 will be released to media, and E. coli strains which does not have immunity protein will be killed.
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)
Lux pL controlled luxR with lux pR controlled GFP(lva tag).
This BioBrick system is able to notify the downstream activation of the lux R promoter of the 3OC6HSL Receiver Device.
The original R0063 BioBrick part (promoter) of the F2621 BioBrick device turned out to be absent in our construct, as was shown by a sequence analysis. To increase the transcription activation there has been put an extra R0063 BioBrick part promoter in front of the F2621 BioBrick device.
The lux p promoters originate from the quorum sensing ability of ''Vibrio fischeri''.
ToxR' aa's 1-210 transmembrane (inner membrane) dimerizing protein. Used in conjunction with J07007-ctx_promotor for dimerization controlled gene expression. aa's 1-210 (abridged from 1-294) removes the w.t. periplasmic region of the protein with intent to replace region with specific binding molecules.
Odr-10(diacetyl)GPCR with optimized codon
Junqi kuang, Xihao liao and Jianhui gong
Odr-10(diacetyl)GPCR encodes a diacetyl-sensing G-protein coupled receptor, originating from the C. elegans. If BBa_K1072010 was transformed into the yeast, the pheromone receptors will be actived and coupled to a heterotrimeric G protein, Odr-10. pathway is composedof Gpa1p (Gα), Ste4p (Gβ) and Ste18p (Gγ) subunits.The Gβγ particle transmits the signal to a MAP kinase module and finally follow a inducible promotor FUS1, which will link a reproter, such as GFP, His3 or LacZ.
As discription of most paper, RNA of Odr-10 be used as plate and transform into receptor cells to express member protein successfully.
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.
Plasmid containing LuxO D47A (pJAF544) was obtained from Bassler lab (Freeman and Bassler, 1999).
Jeremy A. Freeman and Bonnie L. Bassler. A genetic analysis of the function of LuxO, a two-component response regulator involved in quorum sensing in Vibrio harveyi. 1999a. Molecular Microbiology. 31(2), 665-677.
The Cin R (LVA+) protein can be used to sense 3OC14HSL in the media as part of a receiver device
PgolTS-golS-PgolB-rbs-luxI-Ter-Pcons2-rbs-luxR-Ter-Prlux-rbs-OMPA-golB-rbs-luxI-Ter It is a test of our positive feedback circuit for gold ions.
PgolTS-golS-PgolB, luxI, Pcons2, luxR, Prlux, OMPA-golB, Ter
LuxR Protein Generator Controlled by the Left Lux Promoter
This is the BioBrick version of the natural LuxR expression device.
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
Autoinducer inactivation enzyme regulated by LuxR/HSL promoter
We made this construct with LuxR/HSL promoter R0062, B0034 RBS, and the C0060 aiiA gene in order to give us more control with our repressilator system. The aiiA gene will be turned on by the LuxR/HSL promoter in the presence of the LuxR activator protein complexed with the autoinducer 3-oxo-hexanoyl-HSL.
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.
aiiA (LVA-) protein generator driven by plac
Produces aiiA protein from the lac promoter. The aiiA is untagged and should be capable of degrading any intracellular AHL.
Plasmid in positive feedback verification: Pcons2-rbs-lacI-Ter-Plac-rbs-luxI-Ter-Pcons2-rbs-luxR-Ter-Prlux-rbs-rfp-rbs-luxI-Ter It is a positive feedback plasmid using luxI as key to feedback. Introducing IPTG will produce rfp and luxI, and luxI will work back to enhance production of rfp.
Prlux-rbs-rfp-rbs-luxI-Ter. It produces rfp and luxI when there is enough luxR an AHL.
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.
The rpfC gene from Xanthomonas campestris
This is the coding sequence for the RpfC(regulation of pathogenicity factor C) protein. It is the sensor in the two-component signal transduction of Xcc that activates the regulator RpfG(Bba_K1315003) in the presence of DSF.
The sequence had 1 EcoRI site and 3 PstI sites that were all mutated out by site-directed mutagenesis.
The source of this part is ''Xanthomonas campestris pv. campestris''(Xcc) genomic DNA which was provided by the Robert Ryan lab in the Molecular Microbiology department of the University of Dundee, UK.
lasI: autoinducer synthetase for PAI from Pseudomonas aeruginosa. Strong RBS (efficiency=0.6).
Barry Canton [bcanton@mit.edu] and Anna Labno [labnoa@mit.edu]
This device senses the 3OC6HSL concentration in the media and produces output PoPS according to the transfer function given below. LuxR production is controlled by the tet repressible R0040. Initial characterization work has been done on this device using I13273 as a test device.
Example Data Sheets for F2620
Plac-rbs-luxI-Ter. It produces luxI when there is enough IPTG or less lacI.
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.
YFP Producer Controlled by 3OC6HSL Receiver Device
This device is composed of a pLuxL promoter drivinga 3OC6HSL receiver device. The output PoPS is used to produce YFP The YFP should be lightly tagged. This is the exact receiver in the MIT SBC04 synchronization device. It is identical to the previously characterized I13263 but with a tagged output device
single chain Antibody fragment selected to bind to fluorescein molecules. This scFv is the weakest of the three specified sequences. DNA insert in the pCT-4-4-20 (also known as pcT-302) from NheI to XhoI
Substantial Energetic Improvement with Minimal Structural Perturbation in a High Affinity Mutant Antibody
Composite part based on the A340620 PoPS amplifier. pLuxL promoter driving luxR-based HSL receiver. pLux-R is driving a YFP output device.
autoinducer inactivation enzyme aiiA (no LVA)
LuxR and aiiA express constantly. AiiA inactivate AHL as signaling molecule. If AHL concentration exceed the capacity of aiiA, GFP expresses.
this part could accept AHLs and also could produce mRFP easily to detect
ligate the mRFP producer and AHL acceptor together
This device receives AI-1, produces LasR, and reports its production with GFP
comes from existing parts in the Registry (S03966 + S03956)
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.
Lux based Sender/Receiver/Reporter system
Analogue of the V. Fischeri quorum-sensing system. Built using Biobrick components. Includes LuxR and LuxI under the control of their natural promoters and includes a YFP output device on the same transcript as LuxI
This biosensor is constructed to sense C-4 AHL molecules through a method different from BBa_K1157012. This time when AHL molecules are sensed, it produces Rhl receptors which initiates production of CI. Therefore, CI inhibits pCI and the expression of reporter mCherry. When compared to control group, the results will be expected to express lower fluorescence level when there is existence of AHL molecules.
The parts are built by the original biobricks provided by iGEM official, and there are no restriction enzyme sites in the inner part of the construct.
This is built by the biobricks provided by iGEM official
accept AHL signal to produce RoPS and could also produce mRFP
could be easily connect to a protein coding region with a terminator
MC7-rbs-luxI-Ter If there are microcystin, the luxI will be produced.
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.
a fusion protein consisting of E.coli endogenous SOS system repressor LexA and fungus N.crass photosensor protein VVD with LexA carrying mutation at position 40-42 and VVD carrying mutation N56K, C71V and I135M. This part serves as an ultra sensitive photoreceptor(which can sense light as weak as moonlight) and will induce a light-dependent repression of genes with their promoter containing mutated 408 form of SOS box.
LexA carrying mutation at position 40-42 and VVD carrying mutation N56K, C71V and I135M enable the sensor recognize 408 form of SOS box and with enhanced performance.
Lux-based Sender/Receiver Device which outputs PoPS
BioBricked version of Natural Lux system but has no terminator - allows addition of cis elements
lsrR coding gene with ribosome binding site B0034.
pGAL1 + Flag + odr-10 + GFP + ADH1Te + pFUS1 + BFP + ADH1Te
3OC6HSL Receiver Device with Inducible Control of LuxR and a YFP Output device
3OC6HSL receiver with a YFP output device. Same as I13034, but with a TetR regulator under the control of Plac (R0010)
aiiA Protein Generator (LVA+) Controlled by a tet Repressible Promoter
Outputs varying levels of aiiA as part of HSL/aiiA measurements. Subpart of I13205 Formerly known as I13204
LuxS is a synthase for the AI-2 quorum sensing system. This protein is involved in the production of small molecules that are readily absorbed by multiple species of bacteria. LuxS can be used as part of a positive feedback loop in cell to cell signaling.
Nucleotide position 54 changed from A to T in order to destroy a PstI site; this is a sense mutation Also changed stop codon from TAG to TAA (recommended in Registry Help)
The LuxS sequence was pulled from E. coli MG1655 by predesigned primers.
autoinducer synthetase for N-butyryl-HSL (BHL) and HHL
Debra Lin, Srini Devadas, David Gray, Ronny Krashinsky, and Chris Zheng Liu, Boopers
Autoinducer synthesis protein that produces N-butyryl-HSL which binds to RhlR, obtained from Pseudomonas aeruginosa.
Editing Check (1/28/04) (0) BB sites cleaned (1) ATG (2) TAATAA (3) Blast checks out (i.e., it's RhlI) (4) Codon changes OK (AA and tRNA usage) (5) Chassis might have conflict (w/ native autoinducer system?) (6) ssrA tag added (7) barcode added (8) Codon optimized for expression in enteric bacteria 2 silent point mutations have been included into the sequence at base 138 from A to G and at base 576 from G to C to remove internal EcoRI and PstI sites. Mutations were chosen to yield the same amino acid and codons of similar frequency in E. coli.
Reporter tripped by CinR-HSL Threshold, tripped by Lux-HSL Threshold. CinR-HSL also promotes production of aiiA. This should break the thresholds and reset the system.
Elowitz RBS. RhlR CDS (no LVA tag) and terminator. Same as I0466 but no LVA tag
Lux pL controlled LuxR + lux pR autoinducing LuxI (lva tag) + lux pR controlled GFP(lva tag).
Elowitz RBS. CinR CDS (no LVA tag) and terminator. Same as I0468 but no LVA tag
BioBricked version of the natural Lux system with order reversed
Places both LuxI and LuxR under the control of their natural promoters but reverses the order relative to I13211
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
The BCAM0228 Response Regulator from Burkholderia
This is the coding sequence of the BCAM0228 two-component regulator of the BDSF sensory BCAM0227-BCAM0228 system. This system is found in Burkholderia spp.
The sequence had 1 EcoRI site that was mutated out by site-directed mutagenesis.
The source of this part is Burkholderia cenocepacia J2315 genomic DNA which was provided by the Robert Ryan lab in the Molecular Microbiology department of the University of Dundee, UK.
A part designed to emit AHL when given aTc. Uses R0040 and F1611.
Barnase and GFP is co-expressed by luxR/AHL complex.
LuxO D47E, Vibrio harveyi is a constitutively active mutant of LuxO
The plasmid containing LuxOD47E (pJAF822) was obtained from Bassler lab (Freeman and Bassler, 1999). Jeremy A. Freeman and Bonnie L. Bassler. A genetic analysis of the function of LuxO, a two-component response regulator involved in quorum sensing in Vibrio harveyi. 1999a. Molecular Microbiology. 31(2), 665-677.
>gi|156972381:c2983472-2982069 Vibrio harveyi ATCC BAA-1116 chromosome I, complete sequence Nucleotide number 141 has been mutated from T to G, thus bases 139-141 have been changed from GAT (Aspartic acid, D) to GAG (Glutamine, E). Jeremy A. Freeman and Bonnie L. Bassler. A genetic analysis of the function of LuxO, a two-component response regulator involved in quorum sensing in Vibrio harveyi. 1999a. Molecular Microbiology. 31(2), 665-677.
This part is the one of the responding systems of the receiver device of our Photocommunication system
We used ncbi when we couldn't find sequence
EPIC Luciferase: BBa_K325100, Cambridge 2010 pOmpC: BBa_R0082
Pgad - Intracellular Chloride-Sensing Cassette
Pgad is chloride-sensitive promoter which was first discovered in Lactococcus lactis1, which is a gram-positive bacterium which can live in acidic environment. In this operon, gadC is glutamate-gamma-aminobutyrate antiporter and gadB is glutamate decarboxylase. They are both involved in intracellular pH regulation and co-expressed in the same operon under the control of Pgad2. The gene before Pgad, named gadR, which is constitutively expressed under the control of PgadR, is a positive regulator of Pgad coupled genes while intracellular chloride is level elevated.
Synthesized part by GenScript,all the illegal sites for RFC 10 (EcoRI, XbaI, SpeI, PstI) have been substituted by other codon producing the same amino acid in E. coli.
The gene sequence was obtained from the cassette from bp 821 to 2071 of GenBank sequence AF005098, which includes PgadR, gadR, Pgad and the starting codon ATG.
LuxR receiver circuit with an inducible promoter controlling aiiA production downstream. Useful to measure the ability of aiiA (LVA+) to turn off a pLuxL driven receiver circuit in the presence of exogenous HSL
37℃ induced RBS + tetR + double terminator
It is a transcriptional unit including 37℃ induced RBS + tetR + double terminator. It can be added various promoter to adapt your conditions.
To regulate following genes by temperature. To maintain cells at 37?? C ↑ : the tetR protein be generated and repress Ptet's circuit. To release cells at 37?? C ↓ : the tetR protein not be generated and do not repress Ptet's circuit.
the 37℃ induced RBS cames from iGEM08_TUDelft
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
Ser H101 Ala the mid-strength of the 3 piece anti-flour escein scFv sequence set.
cinI codes for an autoinducer synthetase which utilizes SAM to make O3-C14:1-HSL. In complex with O3-C14:1-HSL, CinR (BBa_C0077) 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: tga -> tAaTAA
LasR coding region and a double terminator
The biobricks BBa_C0079 and BBa_B0015 where used for this biobrick.
Luminesensor repressible RecA408 promoter+CheZ
CheZ coding sequence ligated to the Luminesensor repressible recA408 promoter
CheZ is got from E.Coli strain MG1655 by colony PCR
This is the coding sequence for AgrD, the gene encoding the peptide precursor to Autoinducing Peptide, AIP. The AgrD protein product is co-translationally exported, cleaved, and circularized via the AgrB membrane exporter. It then binds to the extracellular domain of AgrC initiating a signalling cascade within the cell.
We wanted to use the coding sequence without the E. coli RBS present in BBa_I746001 and so PCRed this part out. It can be used with any RBS, including gram-positive compatible ones such as BBa_K090505.
This device senses the 3OC6HSL concentration in the media and produces output PoPS according to the transfer function given below. LuxR production is controlled by the LuxR controllable R0063.
PoPS->PoPS Amplifier Controlled by 3OC6HSL
LuxR protein generator upstream of pLuxR. Useful device for cell-cell signaling. Expected to require HSL for maximum PoPS out. Putting this device under the control of a promoter produces a receiver device
Lux pL controlled LuxR with lux pR autoinducing LuxI(lva tag)- AHL.
The F2621 BioBrick device contains a lux pL regulated luxR repressor/activator and a lux pR promoter that is linked to an 3OC6HSL, an AHL autoinducer molecule, synthetase. The lux pL promoter yields a weak constitutive downstream expression. When LuxR and 3OC6HSL are produced, the lux pL promoter, however, will be repressed by a complex of the two. Complementary to the lux pL promoter, the lux pR promoter with a complex of luxR and 3OC6HSL will enable enlarged activation of the downstream synthetase gene, thus increase the concentration 3OC6HSL.
The original R0063 BioBrick part (promoter) of the F2621 BioBrick device turned out to be absent in our construct, as was shown by a sequence analysis. To increase the transcription activation there has been put an extra R0063 BioBrick part promoter in front of the F2621 BioBrick device. The resulting construct worked a lot better than the original F2621 - K082014 BioBrick system.
The lux p promoters and the autoinducer producing enzyme originate from the quorum sensing ability of ''Vibrio fischeri''.
The rpfG gene from Xanthomonas campestris
This is the coding sequence for the RpfG(regulation of pathogenicity factor G) protein. It is the regulator in the two-component signal transduction system of Xanthomonas campestris pv. campestris that is activated by the sensor kinase RpfC(Bba_K1315002) in the presence of DSF(diffusible signal factor).
The source of this part is Xanthomonas campestris pv. campestris(Xcc) genomic DNA which was provided by the Robert Ryan lab in the Molecular Microbiology department of the University of Dundee, UK.
single chain Antibody fragment selected to bind to fluorescein molecules. This scFv is the strongest of the three specified sequences.
GFP Producer Controlled by 3OC6HSL (without Promoter)
3OC6HSL receiver device which need a PROMOTER to control the expression of LuxR
PgolTS-golS-PgolB-rbs-luxI-Ter If there are gold ions, the luxI will be produced.
Produces LuxR protein which can sense 3OC6HSL in the media and activate transcription from R0062, the right hand Lux promoter
The luxR based receiver, I13270, controls the production of the YFP. This construct has been used to characterize I13270.
send P-AI-1 signaling molecules out, using Mnt(or cI) protein repress the pMnt/lac( or plux/cI) hybrid promoter
Receiver circuit. LuxR production is controlled by R0011. Output device is untagged YFP. Because R0011 is stronger than R0040, the switch point of this receiver should be lower than I13273. Built from the following parts - R0011.A340620.E0430
The chimeric GPCR consists of the N- and C- terminals of the rat OR RI7, flanking the TM2-TM7 ligand-binding domain of the c. elegans OR odr-10. It can be functionally expressed in S. cerevisiae, activating the yeast endogenous MAPK pathway upon binding to the ligand diacetyl.
This device senses the 3OC12HSL concentration in the media and produces output PoPS. LasR production is controlled by constitutive P(BIa). This part is the 3OC12HSL analog of F2621.
A part designed to emit AHL when given aTc. Uses R0040 and F1610.
This AHL receiver contains mutant LuxR. it can express GFP 0.1x AHL conc. compared wild type.
Site direct mutagenesis -> Ligation with Biobrick
PoPS in -> luxI out. Strong RBS (efficiency=0.6). Artificial 39 bp terminator (efficiency=0.99).
luxI protein synthesizes 3OC6HSL, which binds to LuxR. Strong RBS (efficiency=0.6).
Prlux-rbs-rfp-rbs-luxR-Ter. It produces rfp and luxR when there is enough rfp and luxR.
PoPS->PoPS amplifier (CinR-based, HSL driven)
CinR protein generator upstream of pCin. Useful device for cell-cell signaling. Expected to require HSL for maximum PoPS out.
autoinducer synthetase for PAI from Pseudomonas aeruginosa
coding region for lasI protein, which produces the chemical signal AI-1
Anti-fluorescein scFv (4M5.3, 15 point mutations) with transcriptional terminator.
This gene sequence includes an EpCAM (Epithelial Cell Adhesion Molecule) antigen binding domain, OmpA transmembrane protein (dock) and TEV protease C terminus. It is planned to use this subpart with BBa_772, which contains only TEV protease N terminus differently instead of C terminus, in order to induce a signal reaction in the case of EpCAM presence. EpCAM is an natural antigen which is found high significantly in the cases of some breast and colorectal cancer subtypes. On the upstream region of protein sequence, there are a constitutive pLac promoter and appropiate RBS. To see how this part works: Experience.
GFP and Producer Controlled by 3OC6HSL Receiver Device
The luxR based receiver, F2622 (formerly I13275 and I13280), controls the production of GFP. The GFP protein generator is the same as that found in I7101.
rhlR repressor/activator from P. aeruginosa PA3477 (+LVA)
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
LuxR receiver circuit (driven by ptet) with pBad controlling the expression of aiiA. Useful for measuring the ability of aiiA (LVA+) to turn off the response of a tet driven receiver cell to exogenous HSL levels
GFP Producer Controlled by 3OC6HSL Receiver Device
The luxR based receiver, F2620 (formerly I13270), controls the production of GFP. The GFP protein generator is the same as that found in I7101.
Biobricked version of the natural Lux quorum sensing system
This device includes a positive feedback loop where LuxI production produces 3OC6HSL that binds to LuxR and activates transcription from R0062 producing more LuxI. There is no output from this device, it must be combined with another device, controlled by R0062 to produce output.
This device senses the 3OC6HSL concentration in the media and produces output PoPS according to the transfer function given below. LuxR production is controlled by the lac repressible R0011.
Constitutive promoter+37℃ induced RBS+tetR+double terminator+Ptet+RBS+GFP+double terminator
We want to utilize a temperature-regulated mechanism to control the expression period of a crystal protein. Therefore, we took advantage of the interaction between 37?? C induced RBS, tetR gene and pTet promoter to create the temperature regulating circuit. The following is a description of our prescribed strategy. First, we use the constitutive promoter, 37?? C induced RBS (K115002), the tetR gene and terminators. We will call this part the ???Regulator???.
Lux pL controlled luxR with lux pR controlled AHL degrading enzyme AiiA(lva tag).
The F2621 BioBrick device contains a lux pL regulated luxR repressor/activator and a lux pR promoter that is linked to an 3OC6HSL, an AHL autoinducer molecule, synthetase. The lux pL promoter yields a weak constitutive downstream expression. When LuxR and 3OC6HSL are produced, the lux pL promoter, however, will be repressed by a complex of the two. If 3OC6HSL is present in the cell, the autoinducer inactivation enzyme A (''aiiA''), with an LVA Tag, will inactivate the 3OC6HSL autoinducer molecules.
autoinducer synthetase for N-butyryl-HSL (BHL) and HHL (no LVA)
Constitutive promoter + 37℃ induced RBS + tetR + double terminator
we took advantage of the interaction between 37?? C induced RBS, tetR gene and pTet promoter to create the temperature regulating circuit. The following is a description of our prescribed strategy. First, we use the constitutive promoter, 37?? C induced RBS (K115002), the tetR gene and terminators. We will call this part the ???Regulator???. We speculate that when the ???Regulator??? and ???Production??? sections are combined, we can achieve a high bacteria growth rate and close to no target protein producing rate at T>37??C, and low bacteria growth rate and high target protein producing rate at T<37??C.
To regulate following gene by temperature
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. .
Anti-fluorescein scFv (4-4-20, no mutations) with transcriptional terminator.
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.
A slightly different sequence than the original; the coverage has been shifted. It should function the same way the original was intended, left repression by hsl-bound LuxR and right activation by hsl-bound LuxR.
autoinducer synthetase for PAI from Pseudomonas aeruginosa (no LVA)
Lux pL controlled LuxR + lux pR promoter.
This part was made as a replacement for a defective version of BBa_F2621 which lacked the R0063 promoter, but was otherwise intact and functional. It shows a significantly higher response to 3OC6HSL than the incomplete BBa_F2621 (which is present in the Registry as BBa_K317037)
Was constructed by cloning BBa_R0063 in front of the incomplete BBa_F2621 (sent from the Registry in July 2011 lacking the R0063 subcomponent)
An enhanced lacI protein generator, inhibits Plac perfectly
This is an enhanced lacI generator. Induced point mutations shifted the structure so it can stop transmitting more perfectly.
Several point mutations were made while this works the best.
A graduate essay published in Zhejiang University, China. Sequence is open to the public.