See BBa_K811003 for details. INPNC with a MCS on C terminus with intervening GS linker domain for surface display of large proteins.
Promoter (luxR & HSL regulated -- lux pR)
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr
Promoter activated by LuxR in concert with HSL The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes.This expression is up-regulated by the action of the LuxR activator protein complexed with the autoinducer, 3-oxo-hexanoyl-HSL. Two molecules of LuxR protein form a complex with two molecules of the signalling compound homoserine lactone (HSL). This complex binds to a palindromic site on the promoter, increasing the rate of transcription.
This promoter is based on the Vibrio fischeri quorum sensing gene promoters. Two genes LuxI and LuxR and transcribed in opposite directions as shown below. The original sequence from which the parts <bb_part>BBa_R0062</bb_part> and <bb_part>BBa_R0063</bb_part> were derived is shown in the picture below. <img src="<bb_file>Image1.gif</bb_file>" width="614" height="362">
An RNA thermometer that theoretically switches on translation at 32 degrees Celcius and switched of translation below that temperature. Still to be tested.
Based on ROSE RNA thermometer sequences from Rfam database.
The MoClo format of BBa_J23104 with AB fusion sites.
This is the MoClo formatted part of BBa_R0079 with EB fusion sites. See the BBa_R0079 page for full information on the part. Summary of modifications from original part: Backbone has one added SpeI site in front of gene, BsaI sites, and 4bp fusion sites.
Proteins C and D from the acs cellulose producing operon of Gluconacetobacter Xylinus
Protein coding sequence from genome of Gluconacetobacter Xylinus, resynthesized and codon optimised for E. coli
Contains an IPTG inducible promoter an RBS, RFP, and terminator.
Produces LuxR protein which can sense 3OC6HSL in the media and activate transcription from R0062, the right hand Lux promoter
This part is designed as a temperature inducible RBS. Only designed so far.
The secondary structure is important to the function of these regions, but part of the wt secondary structure is destroyed by the scar. We've tried to alter the sequence so the predicted structure (through mfold and those kind of servers) is sort of conserved, but temperature sensitivity still has to be tested. If it doesn't work, possible solution might be the addition of a larger conserved part of the wt, which implies a small part of wt protein sequence as well.
This sequence is taken from the Bradirhizobium Japonicum (BA000040.) as the 5'UTR (ROSE) of a heat shock protein (prfA).
Predicted Stem Loop extending to startcodon
This sequence is taken from the Listeria Monocytogenes (EU372032.). It's the 5'UTR of a TF induced at 37 degrees.
Promoter, Standard (luxR and HSL regulated -- lux pR)
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr Drew Endy
[Note: This is the same part as R0062 except that the -10 and -35 sites and spacing have been changed to comply with BBa_S0001]. Promoter activated by LuxR in concert with HSL The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes.This expression is up-regulated by the action of the LuxR activator protein complexed with the autoinducer, 3-oxo-hexanoyl-HSL. Two molecules of LuxR protein form a complex with two molecules of the signalling compound homoserine lactone (HSL). This complex binds to a palindromic site on the promoter, increasing the rate of transcription.
This promoter is based on the Vibrio fischeri quorum sensing gene promoters. Two genes LuxI and LuxR and transcribed in opposite directions as shown below. The original sequence from which the parts <bb_part>BBa_R0062</bb_part> and <bb_part>BBa_R0063</bb_part> were derived is shown in the picture below. <img src="<bb_file part=BBa_R0062>Image1.gif</bb_file>" width="614" height="362"> Modified to comply with BBa_S0001: TTGACA-17N-GATACT
MoClo format of a modified version of BBa_B0030 with BC fusion sites.
This is the MoClo formatted part of a modified BBa_B0030 with BC fusion sites. See the BBa_R0010 page for full information on the part. This is a Level 0 MoClo part with flanking sites B on the 5' side and site C on the 3' side of the sequence. The fusion site letters refer to 4bp fusion sites: A = GGAG; B = TACT; C = AATG; D = AGGT; E = GCTT; F = CGCT; G = TGCC; H = ACTA. This part is in a MoClo backbone that is a modified version of the pSB1C3 backbone. See <a href: http://parts.igem.org/Part:BBa_B0030???>BBA_K783058 Summary of modifications from original part: Backbone has one added SpeI site in front of gene, BsaI sites, and 4bp fusion sites.
This part codes ChiA1 and its RBS. Its start codon is not AUG but GUG.
T7 promoter for expression of functional RNA
Built by Josh as an intermediate in screening plasmid construction.
gfasPurple, purple chromoprotein (incl RBS, J23110)
This chromoprotein from the coral Galaxea fascicularis, gfasPurple, naturally exhibits strong color when expressed.
Synthesized and codon optimized for E coli by GenScript.
mmoB is one subunit of the mmo from Methylococcus capsulatus bath
the brick is constructed in the RCF10 standard
Amplified from the genomic DNA of Methylococcus capsulatus bath
Complement of Ethanolamine Utilization Microcompartment proteins from Salmonella LT2
Matthew Adams, Swati Choudhary, Anthony Goering, Rachel Farr, Ethan Johnson, Annie Kathuria, Poonam Srivastava, Ian Windsor
Three proteins subunits of a bacterial micro-compartment. The genes are in the pSB1C3 plasmid. A total of 5 micro-compartment genes are cloned in a single plasmid. They are arranged as two sets of two genes (in a single operon) with one gene which is upstream to the pairs. They are flanked directly upstream by a mutated Lac promoter to make them constitutively expressed. The genes were amplified by PCR from genomic DNA using primers that would introduce restriction sites flanking the genes.
The genes were amplified by PCR from genomic DNA using primers that would introduce restriction sites flanking the genes. The genes were ligated into a modified pUC biobrick vector containing a constitutive promoter. The genes were taking in their native operons and ligated sequential according to size.
This regulatory part is made up of the Arabinose-Inducible promoter, pBAD, and its transcriptional inhibitor/activator, AraC. AraC inhibits gene expression in the absence of Arabinose by binding to the operator units present within the AraC and pBAD promoter sequences, and producing a DNA loop that prevents the RNA polymerase and CAP from binding and initiating transcription. When Arabinose is supplied, this binds to the AraC dimer and releases it from the operator site found in the pBAD promoter, at the same time it enhances transcription by allowing binding to the enhancer sites upstream.
This part was cloned out of existing templates (such as BBa_K325218, BBa_325219, all of which contain sfGFP downstream of the pBAD promoter)
tsPurple, purple chromoprotein (incl RBS, J23110)
This chromoprotein naturally exhibits strong color when expressed.
Cloned from TinselPurple vector, DNA 2.0.
This part accepts POPS to generate EYFP under the medium strength RBS B0032.
The MoClo format of BBa_R0040 with AB fusion sites.
===Design Notes=== This is the MoClo formatted part of BBa_R0040 with AB fusion sites. See the BBa_R0040 page for full information on the part. This is a Level 0 MoClo part with flanking sites A on the 5' side and site B on the 3' side of the part. The fusion site letters refer to 4bp fusion sites: A = GGAG; B = TACT; C = AATG; D = AGGT; E = GCTT; F = CGCT; G = TGCC; H = ACTA. Summary of modifications from original part: Backbone is a modified version of pSB1C3 with added SpeI site in front of gene, BsaI sites flanking, and 4bp fusion sites. See BBa_K783055 . ===Source=== iGEM Distribution Kit ===References===
Protein coding region for the NanoLuc(tm) Luziferase (designed by the Promega Corporation, free for research use), Codon optimized for yeast. The Nanoluc is smaller than other luziferase proteins and yields equal or stronger luminescence readings.
gBlock designed by Team Tuebingen, sequence by Promega
AraC operator, truncated to include araO1, araI1, araI2, c-amp1, and c-amp2 sites. This operator should *activate* transcription in the presence of AraC; b/c the operator lacks the araO2 site, there should not be araC-mediated repression.
Binding region for LasR protein (positive regulation)
"Analysis of the Pseudomonas aeruginosa Elastase (lasB) Regulatory Region". Lynn Rust, Everett Pesci, and Barbara Iglewski
Promoter (lambda cI and luxR regulated -- hybrid)
cI repressor negatively regulates this promoter and LuxR activates its transcription.The effect of cI is dominant over LuxR. This part is based on the LuxR and cI repressor regulated hybrid promoter designed and tested by Ron Weiss. It requires the binding of two cI repressor dimers for maximal repression and contains two cI repressor binding sites namely, OR1 and OR2. This promoter is leaky in the sense that 'some' transcription is seen in the absence of both cI and LuxR. LuxI cI activity of promoter + + zero + - maximum - + zero - - leaky (no quantitative information)
This part was designed based on the LuxR and cI repressor regulated hybrid promoter tested by Ron Weiss and the LuxR-LuxICDABE sequence annotated by Tom Knight AF170104 .
Contains both a BbsI and a BstXI restriction site. When cut with both enzymes, it leaves an EcoRI and a PstI site.
70 bp from Eco to Spe or Xba to Pst, to allow for gel extraction. The middle portion is largely noise, intended as filler to raise the size of the part.
Promoter (luxR & HSL regulated -- lux pL)
The lux cassette of V. fischeri contains a left and a right promoter. The left promoter gives weak constitutive expression of downstream genes.This expression is down-regulated by the action of the Lux repressor, LuxR. Two molecules of LuxR protein form a complex with two molecules the signalling compound homoserine lactone (HSL). This complex binds to a palindromic site on the promoter, increasing the rate of transcription from Pr, repressing transcription from Pl
This promoter is based on the Vibrio fischeri quorum sensing gene promoters. Two genes LuxI and LuxR and transcribed in opposite directions as shown below. The original sequence from which the parts <bb_part>BBa_R0062</bb_part> and <bb_part>BBa_R0063</bb_part> were derived is shown in the picture below.Includes most of Lux reulatory region, including the LuxR binding site which activates the right promoter. A putative LuxR autorepression binding site is also identified adjacent to the -10 site of the right promoter. This 2nd site has 55% identity with the first site. Putative inverted repeats (of size 18-27 bp) also exist between these two sites (not marked above), which may represent binding sites for other regulatory proteins. <img src="<bb_file>Image01.gif</bb_file>" width="614" height="362"> Unspecified.
Ralf Beer, Konrad Herbst, Nikolaos Ignatiadis
The IndC Indigoidine Synthetase from Photorhabdus luminescens laumondii TT01 is a nonribosomal peptide synthetase (NRPS) which produces the blue pigment indigoidine. Indigoidine itself consists of two cyclic glutamines. IndC is a single module NRPS and consists of an Adenylation-, Thiolation- and a Thioesterase-domain. The A-domain thus contains an internal Oxidation domain.
The native indC contains an internal EcoRI and SpeI cutting site which was mutated using a CPEC approach.
The coding sequence was amplified from Photorhabdus luminescens laumondii TT01 DSM15139.
Nuclease from Staphylococcus aureus. Sequence pending.
dxs coding sequence encoding 1-deoxyxylulose-5-phosphate synthase
This is the coding sequence of dxs from Escherichia coli JM109. It encodes 1-deoxyxylulose-5-phosphate synthase, thiamine-requiring, which catalyses teh first step in the biosynthesis of terpenoids. Overexpression of dxs has been reported to increase yields of carotenoids and other terpenoids [add reference here].
CinR (BBa_C0077) in complex with O3-C14:1-HSL (from CinI, BBa_C0076) binds to this promoter and activates transcription. This promoter contains the RBS: TGGAGG
The regulatory locus cinRI in Rhizobium leguminosarum conrols a network of quorum-sensing loci Lithgow, JK; Wilkinson, A; Hardman, A; Rodelas, B; Wisniewski-Dye, F; Williams, P; Downie, AJ MOL. MICROBIOLOGY 37(1): 81-97, 2000 Change log: the last 5 bases (gctaa) removed to allow for the correct spacing between the RBS (TGGAGG) and the start of the downstream gene after biobricks splicing.
unidentified/uncharacterized CinR dependent
N-terminal half of TEV Protease S219V mutant (for Split-TEV-Protease applications) in RFC[25]
N-terminal half of the TEV Protease (autolysis resistant S219V mutant) can be used for Split-TEV-Protease applications where autolysis of the reconstituted TEV Protease is not wanted. The necessary C-terminal half can be found under BBa_K1159102. This part is flanked by RFC[25] pre- and suffix for further protein fusions.
The MoClo format of BBa_J23116 with AB fusion sites.
===Design Notes=== This is the MoClo formatted part of BBa_J23116 with AB fusion sites. See the BBa_J23116 page for full information on the part. This is a Level 0 MoClo part with flanking sites A on the 5' side and site B on the 3' side of the part. The fusion site letters refer to 4bp fusion sites: A = GGAG; B = TACT; C = AATG; D = AGGT; E = GCTT; F = CGCT; G = TGCC; H = ACTA. Summary of modifications from original part: Backbone is a modified version of pSB1C3 with added SpeI site in front of gene, BsaI sites flanking, and 4bp fusion sites. See BBa_K783055 . ===Source=== iGEM Distribution Kit ===References===
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.
Vinay S Mahajan, Brian Chow, Peter Carr, Voichita Marinescu and Alexander D. Wissner-Gross
The cI regulated promoter is based on the pR promtoer from bacteriohage lambda. The promoter has two two DNA binding sites for lambda cI repressor <bb_part>BBa_C0051</bb_part>. cI binding results in repression of transcription. The specific sequence used here is based on the cI repressible promoter used in the Elowitz repressilator (and references therein).
In order to address concerns about the promoter transcribing in the reverse direction, we have removed the -35 and -10 signals responsible for the promoter activity in the reverse direction. (More details needed here! DE, 2/24/03) Incompatible with host expressing cI repressor.
A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000
Tat secretion cassette with constitutive promoter.
This cassette allow E.coli to secrete proteins with torA signal. Wild type Tat protein secretion system is too week so that Kyoto 2012 construct Tat cassette to reinforce the ability of transportation of Tat system. This parts include tatA,B,C protein coding region and pspA (phage shock protein A). Tat A,B,C protein is the main component of Tat complex where proteins with torA signal go through and pspA can encourage protein secretion via Tat system. Kyoto 2012 suggest iGEMers with this new way of secretion and provide them with this cassette regulated by constitutive promoter.
This Device has too long sequence to read it so that we read each components' sequence separately and conjugated them to make the information about sequence of the device
tatABCD and pspA are derived from Escherichia coli's genomic sequence
Vincent Leonardo, Christopher Wall and Robert Mckiernan
This BioBrick facilitates the switching of Bacillus subtilis cells between rod-shape to L-form, a cell wall-less form of bacteria. It consists of chloramphenicol acetyl-transferase (cat) gene and a xylose-controlled promoter (PxylR) which are flanked by ~300bp pbpB gene at the 5???end and ~300bp of murE gene at 3???end. These flanking sequences allows the integration of the BioBrick into the chromosome by homologous recombination replacing the region containing the end of pbpb gene and the beginning of the murE gene, which are involved in peptidoglycan biosynthesis. This event replaces the constituitive murE promoter with a PxylR upstream of the murE gene. Therefore, when xylose is present cell wall biosynthesis is switched on, giving rod cells. When xylose is not present, the cells will become L-forms. Simply transform the BioBrick into competent B. subtilis cells and through recombination it will integrate into cells chromosome. Plate out the transformants onto 1.5% lysogeny broth agar containing 5??g/ml Chloramphenicol and 0.8 %( w/v) xylose. This will give you rod cells containing biobrick. To get L-forms, inoculate some colonies into a solution containing an equal amount of nutrient broth and MSM. Also inoculate some transformants into the same media with the addition of 0.8 %( w/v) xylose to clarify that the switch BioBrick is working. Use microscopy to check the morphology of the cells in each media. L-forms will appear near spherical and can vary in size considerably.
There were 3 restriction sites that did not meet the RFC10 requirements, two EcoRI sites and one PstI. The first EcoRI restriction site was located 218bp into the whole BioBrick (inside pbpb region), we changed the sequence from GAATTC to GCATTC. The second restriction site was located 2287bp into the BioBrick in the spacer region between PxylR and murE, we changed it from GAATTC to GTATTC. The PstI site was located 197bp into the BioBrick in the pbpb region and was change from CTGCAG to CTTCAG.
The part is the courtesy of Prof. Jeff Errington research group, and all the sequences come from the Bacillus subtilis genomic sequence. The pbpb section in the BioBrick was taken from end of the pbpb gene 1841-2151bp (309bp). Sequence acquired through microbes online VIMSS38051. The murE section was taken from the start of the murE gene 1-331bp. Sequence from microbes online VIMSS38053. The cat gene and PxylR gene were both come from the B. subtilis genome, but can also be found in the BioBrick Registry (BBa_K823020 and BBa_K143014).
enhanced yellow fluorescent protein derived from A. victoria GFP
This part codes for a promoter that works in both E. coli and L. bacillus. It is characterized agains J23101, when J23101 had 4322 AU, this promoter had 17518 AU.
This part is designed with the same sticky ends as you would get by cutting it with EcoRI-HF and Spel.
This part is a synthetic promoter based on a promoter library from the article "The Sequence of Spacers between the Consensus Sequences Modulates the Strength of Prokaryotic Promoters." by Peter Ruhdal Jensen and Karin Hammer.
Bicistronic design RBS 8, MoClo Format with BC fusion sites
This is the MoClo formatted part of a bicistronic design ribosomal binding site 8 with BC fusion sites. This is a Level 0 MoClo part with flanking sites B on the 5' side and site C on the 3' side of the part. The fusion site letters refer to 4bp fusion sites: A = GGAG; B = TACT; C = AATG; D = AGGT; E = GCTT; F = CGCT; G = TGCC; H = ACTA. Backbone is a modified version of pSB1C3 with added SpeI site in front of gene, BsaI sites flanking, and 4bp fusion sites. See BBa_K783055.
This part was cloned off a BCD template from Sonya Iverson. Sonya Iverson based her designs for her BCD2 part off of a paper published by Mutalik, et al.
Overlap extension of synthetic oligonucleotides
This part is an intermediate to the protein generator BBa_45200.
spisPink, pink chromoprotein (incl RBS, J23110)
This chromoprotein from the coral Stylophora pistillata, spisPink, naturally exhibits color when expressed.
This is a MoClo converted version of BBa_J23110
This is a MoClo converted version of BBa_J23110. It has two MoClo fusion sites flanking this part.
We had to design the proper MoClo fusion sites to flank this part.
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)
TEV protease with anti-self cleavage mutation S219V, codon optimized for E. coli
This part is a TEV protease in RFC25 that was optimized for expression in E. coli. The part contains the S219V anti-self cleavage mutation.
The part does not contain start or stop codons, because it was designed for RFC25 compability
The part was codon optimized using the genomic amino acid sequence, designed for RFC25 and mutated S219V for anti-self cleavage properties.
The MoClo format of BBa_J23103 with AB fusion sites.
amajLime, yellow-green chromoprotein (incl RBS)
This chromoprotein from the coral Anemonia majano, amajLime, naturally exhibits strong color when expressed.
This part is made up of the coding sequence of the A and B elements of the acs, cellulose-producing operon. AcsA and acsB are transcribed and translated in the form of a single polypeptide, and form the main two domains (regulatory and catalytic) of the Cellulose Synthase enzyme in Gluconacetobacter Xylinus.
The Ribosomal binding site of AcsAB is already included within the sequence. Because we are transferring this element from Gluconacetobacter into simpler organisms, such as E.coli, the native RBS was replaced with a new RBS, that would guarantee strong translation whilst maintaining the native stoichiometry for the final operon system to operate successfully.
The AcsAB coding sequence was extracted from NCBI, codon optimised and edited accordingly for further gene synthesis and cloning into pSB1C3.
Promoter (lacI regulated, lambda pL hybrid)
Neelaksh Varshney, Grace Kenney, Daniel Shen, Samantha Sutton
Inverting regulatory region controlled by LacI (<bb_part>BBa_C0010</bb_part>, <bb_part>BBa_C0011</bb_part>, etc.) The PLlac 0-1 promoter is a hybrid regulatory region consisting of the promoter P(L) of phage lambda with the cI binding sites replaced with lacO1. The hybrid design allows for strong promotion that can nevertheless be tightly repressed by LacI, the Lac inhibitor (i.e. repressor) (<bb_part>BBa_C0010</bb_part>) ([LUTZ97]). The activity of the promoter can be regulated over a >600-fold range by IPTG in E.Coli DH5-alpha-Z1 (same paper reference).
hybrid promoter design to create strong promoter that is, at the same time, highly repressible. note that the upstream operator installed in this hybrid is slightly different than the one in the original source (Lutz and Bujard, 1997). the most upstream operator region is slightly truncated in the represillator version, so that both operators in the hybrid are the same sequence. see references for details. also, the sequence has been truncated after the transcriptional start site. LacI binds to this regulator. This part is incompatible with species containing active LacI coding regions. Lactose and IPTG disable the operation of LacI and increase transcription. This part is incompatible with environments containing lactose or lactose analogs.
represillator of Elowitz and Leibler (2000)
Glucose oxidase codon optimised for Synechocystis
Glucose oxidase gene codon optimised for Synechocystis sp. PCC 6803
The p22 cII regulatory region sequence is a 97 base-pair sequence with the standard BioBrick prefix and suffix sections on its ends. p22 cII repressor protein, BBa_C0053, binds to it. This segment contains O-R1, O-R2, a fragment of O-R3, the -35 of P-RM, and P-R (-10 and -35 from Tom Knight)
autoinducer inactivation enzyme aiiA (no LVA)
The CRY2/CIBN interaction is entirely genetically encoded. The binding reverses within minutes in the dark, allowing rapid shutoff of transcription by placing samples in the dark. This fusion protein is for use in LACE(light-activated CRISPR/Cas9 effector) system, and a tCas9 fused to its N terminus. To regulate DNA transcription by blue light, the system is based on CRY2/CIBN interaction in which a light-mediated protein interaction brings together two protein (tCas9 and an activation domain VP64) . If we remove the stimulation of blue light, dark reversion of CRY2 will dissociate the interaction with CIBN and shut off transcription. tCas9 can be tagged with transcriptional activators, and targeting these dCas9 fusion proteins to the promoter region results in robust transcription activation of downstream target genes. This tCas9-based activators is the case that tCas9 fused directly to a single transcriptional activator( VP64).
mutant EcoR 1 : TGTTGGGAATTc--->TGTTGGGAATTg
tCas9 is from GE Share company. VP64 is synthetic construct.
Delta 9 Desaturase, optimised for expression in Escherichia coli
Robert Harrison, Lorna Hepworth, Vun Hiang Tan
This enzyme is obtained fro the gene Stearoly Co-A Desaturase (SCD). The gene is obtained from Synechocystis sp., a cyanobacterium.It is involved in the biosynthesize of C18:1 from C18:0. C18:1 corresponds to Oleic acid. The delta 9 desaturase introduces cis carbon double bond on the 9th carbon in C18:0.
Codon optimized for expression in E. coli
Synthesized gene based off the protein sequence from Synechocystis sp
R6K origin of replication requires a pir+ or pir116 strain for replication in the absence of a second origin. In most E. coli strains, this is a silent feature. {pSB1A2-Bca1011}
PCR ca1011F/R on pG80ko (445 bp, EcoRI/SpeI) Sub into pSB1A2-I13522 (EcoRI/SpeI) Product is Bca1011 ---- ca1011F Forward Biobricking of R6K GGACTgaattcgcggccgcttctagagtgattcgcacgggcccatg ca1011R Reverse biobricking of R6K ccgctactagtaGCAGTTCAACCTGTTGATAG
BS3 is a plant specific promoter which is induced by the TALE AvrBS3. This part is compatible with the GoldenGate MoClo Assembly Standard (free from BsaI and BpiI recognition sequences).
Domestication for GoldenGate (Removal of internal BsaI and BpiI recognition sequences).
Pxyl, a xylose inducible promoter + its regulator XylR
Pxyl is a promoter which is repressed by XylR. In presence of Xylose, XylR leaves the operator sequence and the Pxyl is active. XylR is present in most B. subtilis strains, so this would be a second copy of the gene. XylR is provided with constittive promoter, RBS and terminator.
This is a MoClo converted version of BBa_J23115
This is a MoClo converted version of BBa_J23115. It has two MoClo fusion sites flanking it.
Protein generator converting TIPS to the protein cII from p22. Used as the input section for Quad Part Inverters Q01530.
mCherry (BBa_J06504) compatible with Genabler assembly
mCherry (BBa_J0654) part with inward facing EarI recognition sites at the ends compatible with Genabler assembly.
To be compatible with Genabler assembly, the part has inward-facing EarI sites at the ends. When assembled by BioBrick assembly, five N-terminal residues MetAlaSerSerSer- and one C-terminal Ala residue will be added to the original sequence of BBa_E0040.
mCherry is based on the BioBrick BBa_J06504.
The MoClo format of BBa_J23107 with AB fusion sites.
This is the MoClo formatted part of BBa_J23107 with AB fusion sites. See the BBa_J23107 page for full information on the part. Summary of modifications from original part: Backbone has one added SpeI site in front of gene, BsaI sites, and 4bp fusion sites. The fusion site letters refer to 4bp fusion sites: A=GGAG; B=TACT; C=AATG; D=AGGT; E=GCTT; F = CGCT; G=TGCC; H=ACTA.
The asr promoter is a pH-responsive promoter.
This part contains the asr promoter with its native RBS. The asr promoter is a pH-responsive promoter native to E. coli. It induces transcription in acidic conditions (~pH 5.5).
This is a MoClo converted version of BBa_R0063
This is the MoClo converted version of BBa_R0063, which is the negatively regulated pLuxR promoter. It has two MoClo fusion sites flanking it.
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.
engineered cyan fluorescent protein derived from A. victoria GFP
It is a CDS of PhiC31 integrase from Streptomyces phage. It has a nuclear location sequence in the N-terminus.
It has a nuclear location sequence (NLS) and its codons are optimized for plants.
Collection of promoters regulated by LldR.
Tet operator with CFP reporter (with LVA tag) [R/Tc+]
Christopher Batten, Victoria Chou, Kenneth Nesmith
asPink, pink chromoprotein (incl RBS, J23110)
This chromoprotein from the coral Anemonia sulcata, asCP, naturally exhibits color when expressed.
Bicistronic Design RBS 2, MoClo Format with BC fusion sites
This is the MoClo formatted part of a bicistronic design ribosomal binding site 1 with BC fusion sites. This is a Level 0 MoClo part with flanking sites B on the 5' side and site C on the 3' side of the part. The fusion site letters refer to 4bp fusion sites: A = GGAG; B = TACT; C = AATG; D = AGGT; E = GCTT; F = CGCT; G = TGCC; H = ACTA. Backbone is a modified version of pSB1C3 with added SpeI site in front of gene, BsaI sites flanking, and 4bp fusion sites. See BBa_K783055.
Inducible promoter of the lux quorum sensing system with RBS
This construct is derived from BBa_R0062 and BBa_B0032. It combines the lux induced promoter with an RBS and can be used to clone it in front of a desired gene of interest, which shall be induced by lux.
The construct was combined from BBa_R0062 and BBa_B0032, which were taken from the 2013 distribution kit.
mmoX is one subunit of the mmo from Methylococcus capsulatus bath
Amplified from the genomic DNA of Methylococcus capsulatus bath without any PstI restriction site
Protein generator converting TIPS to the protein cI from lambda. Used as the input section for Quad Part Inverters Q01150 and Q01151.
Lac operator with CFP reporter (without LVA tag) [R/Lc-]
The MoClo format of BBa_J23100 with AB fusion sites.
This is the MoClo formatted part of BBa_J23100 with AB fusion sites. See the BBa_J23100 page for full information on the part.
This is the MoClo formatted part of BBa_J23100 with AB fusion sites. See the BBa_J23100 page for full information on the part. Summary of Modifications from original part: Backbone has one added SpeI site in front of gene, BsaI sites, and 4bp fusion sites.
Nitric oxide reductase (norB) from Pseudomonas aeruginosa PAO1
This part conteins gvpA protein's coding sequence.
This part conteins gvpA protein's coding sequence. This gene from Planktothrix rubescens can be succesfully expressed in E. coli. The gvpA's expression have been examined by another part " J23106+B0034+gvpA+B0015". (Since it was ligated on cloning vector pSB4A5, we didn't register this part. The gvpA protein conteins a typical domain of the superfamily 'Gas vesicle'(NCBI CDD cl02594). This gene is quiet conserved in most gvp's polycistron. According to Anthony E. Walsby's research, gas vesicles have a similar morphology. GvpA protein is a kind of small hydrophobic protein which forms the ribs of the main structure. 'All cyanobacterial gas vesicles so far analyzed contain a protein (GvpA) of about 7.4 kDa that forms the main mass of the structure and must be responsible for many of its properties.' (Gas Vesicles. Microbiological Reviews. A. E. Walsby)
According to Anthony E. Walsby's research, gas vesicles have a similar morphology. GvpA protein is a kind of small hydrophobic protein which forms the ribs of the main structure. 'All cyanobacterial gas vesicles so far analyzed contain a protein (GvpA) of about 7.4 kDa that forms the main mass of the structure and must be responsible for many of its properties.' (Gas Vesicles. Microbiological Reviews. A. E. Walsby)
MoClo format of a modified version of BBa_B0031 with BC fusion sites.
This is the MoClo formatted part of a modified BBa_B0031 with BC fusion sites. See the BBa_B0031 page for full information on the part. This is a Level 0 MoClo part with flanking sites B on the 5' side and site C on the 3' side of the sequence. The fusion site letters refer to 4bp fusion sites: A = GGAG; B = TACT; C = AATG; D = AGGT; E = GCTT; F = CGCT; G = TGCC; H = ACTA. This part is in a MoClo backbone that is a modified version of the pSB1C3 backbone. See <a href: http://parts.igem.org/Part:BBa_K783058???>BBA_K783058 Summary of modifications from original part: Backbone has one added SpeI site in front of gene, BsaI sites, and 4bp fusion sites.
C-terminal half of TEV Protease S219V mutant (for Split-TEV-Protease applications) in RFC[25]
C-terminal half of the TEV Protease (autolysis resistant S219V mutant) can be used for Split-TEV-Protease applications where autolysis of the reconstituted TEV Protease is not wanted. The necessary N-terminal half can be found under BBa_K1159101. This part is flanked by RFC[25] pre- and suffix for further protein fusions.
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.
pCooF Promoter from Rhodospirillum rubrum
CooA is a CO-sensing protein that activates the transcription of genes encoding the CO-oxidation (coo) regulon, whose polypeptide products are required for utilizing CO as an energy source in Rhodospirillum rubrum. CooA binds to a position overlapping the -35 element of the P(cooF) promoter, similar to the arrangement of class II CRP (cAMP receptor protein)- and FNR (fumarate and nitrate reductase activator protein)-dependent promoters when expressed in Escherichia coli. The CO-dependent anaerobic growth of Rhodospirillum rubrum relies on a CO oxidation system encoded by two CO regulated transcriptional units, cooMKLXUH and cooFSCTJ. The key products of the coo regulon are an O2- sensitive CO dehydrogenase (CooS), a CooS-associated Fe-S protein (CooF), and a CO-tolerant hydrogenase (CooH), and the expression of the genes depends upon the activity of the CooA protein, which senses CO under anaerobic conditions. CO-independent basal level transcription of PcooF is not detectable due to the absence of active CooA.
The sequence information was acquired from NCBI and physical DNA was synthesized from GENEART. Classical cloning strategies(restriction digestion) were used to produce biobricks.
S.W. Singer, M.B. Hirst and P.W. Ludden, CO-dependent H2 evolution by Rhodospirillum rubrum: role of CODH:CooF complex, Biochim Biophys Acta - Bioenerg 1757 (2006), pp. 1582???1591
This part accepts POPS to produce ECFP under the medium RBS B0032
amajLime, yellow-green chromoprotein (incl RBS & J23110)
promotor of the fet3 gene from S. cerevisiae, fet3 is repressed on high iron levels.
length of promoter was chosen to include binding sites and to obtain good primers
genomic upstream sequence of fet3 gene from the Saccharomyces Genome Database (http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=fet3)
amilGFP, yellow chromoprotein (incl RBS)
This chromoprotein from the coral Acropora millepora, amilGFP, naturally exhibits strong yellow color when expressed. The color is readily visible to naked eye both in LB-culture and on agar plates. Color development can be seen in less than 24 hours of incubation.
Two illegal internal restriction sites (EcoRI and PstI) were removed.
Acropora millepora. The DNA was provided by Jeffrey Miller at UCLA, and was made BioBrick-compatible by removing two illegal internal restriction sites (EcoRI and PstI).
Jay Blumling, Debra Lin, Madeleine Sheldon-dante, Fred Tan, MIT SMUG
Variant of the wild-type pLuxR promoter with lower sensitivity
BBa_K1216007 is a variant of the wild-type luxR promoter with a lowered sensitivity for LuxR-AHL.
LuxR binding site were mutated to reduce the sensitivity of the promoter.
The promoter was built through site-saturation mutagenesis of the wild-type promoter.
The MoClo format of BBa_J23113 with AB fusion sites.
===Design Notes=== This is the MoClo formatted part of BBa_J23113 with AB fusion sites. See the BBa_J23113 page for full information on the part. This is a Level 0 MoClo part with flanking sites A on the 5' side and site B on the 3' side of the part. The fusion site letters refer to 4bp fusion sites: A = GGAG; B = TACT; C = AATG; D = AGGT; E = GCTT; F = CGCT; G = TGCC; H = ACTA. Summary of modifications from original part: Backbone is a modified version of pSB1C3 with added SpeI site in front of gene, BsaI sites flanking, and 4bp fusion sites. See BBa_K783055 . ===Source=== iGEM Distribution Kit ===References===
Constitutively expressed chromoprotein eforRed
The chromoprotein eforRed is constitutively expressed and can be used for the marking of cells for easier differentiation.
This part is a combination of BBa_K1073006 and BBa_K592012, which were derived from the 2013 distribution kit and the iGEM Team Uppsala.
Jou-Chien Liao, Rohan Shina, Yucheng Huang
double terminator (B0012-B0011), reversed
Constitutively expressed chromoprotein amilGFP
The chromoprotein amilGFP is constitutively expressed and can be used for the marking of cells for easier differentiation.
This part is a combination of BBa_K1073006 and BBa_K592010, which were derived from the 2013 distribution kit and the iGEM Team Uppsala.
MoClo formatted version of B0033 with BC fusion sites
This is the MoClo formatted part of BBa_B0033 with BC fusion sites. See the BBa_B0033 page for full information on the part. This is a Level 0 MoClo part with flanking sites B on the 5' side and site C on the 3' side of the part. The fusion site letters refer to 4bp fusion sites: A = GGAG; B = TACT; C = AATG; D = AGGT; E = GCTT; F = CGCT; G = TGCC; H = ACTA. Summary of modifications from original part: Backbone is a modified version of pSB1C3 with added SpeI site in front of gene, BsaI sites flanking, and 4bp fusion sites. See BBa_K783055.
The MoClo format of BBa_J23118 with AB fusion sites.
===Design Notes=== This is the MoClo formatted part of BBa_J23118 with AB fusion sites. See the BBa_J23118 page for full information on the part. This is a Level 0 MoClo part with flanking sites A on the 5' side and site B on the 3' side of the part. The fusion site letters refer to 4bp fusion sites: A = GGAG; B = TACT; C = AATG; D = AGGT; E = GCTT; F = CGCT; G = TGCC; H = ACTA. Summary of modifications from original part: Backbone is a modified version of pSB1C3 with added SpeI site in front of gene, BsaI sites flanking, and 4bp fusion sites. See BBa_K783055 . ===Source=== iGEM Distribution Kit ===References===
enhanced cyan fluorescent protein derived from A. victoria GFP
Bidirectional transcriptional terminator consisting of a 22 bp stem-loop.
In the naturally-occuring sequence there is a mismatch in the stem of the stem loop. This can be corrected via an A->G mutation (at position 40 -- sequence coordinate/not MFOLD coordinate). The above sequence does not reflect this mutation (but the MFOLD image does). This terminator's location cannot be found using some inverted repeat detectors like PALINDROME because it is too short and contains a mismatch. This one was found with the help of Tom Knight. It lies between two coding regions that point towards eachother.
Derived from luxICDABEG operon terminator of Vibrio fischeri AF170104 .
Benzalacetone Synthase, compatible with MoClo
SV40NLS-PhyB-linker-LexA (part for a LexA based light-switchable promoter system)
Composite part of BBa_K801030, BBa_K801030, BBa_K365005, BBa_K801033
Essential Gly for photoconvertible binding of PIF3
Essential Arg for photoconvertible binding of PIF3
SV40 nuclear localization sequence (recognized by importin alpha)
Essential Cys for chromophore attachement of phycocyanobilin (PCB)
Chloramphenicol resistance gene including its native promoter and ribosome binding site. Confers resistance to 25 ug/mL chloramphenicol. {pSB1A2-Bca1016}
PCR ca1016F/R on pKD3 (934 bp, EcoRI/SpeI) Sub into pSB1A2-I13522 (EcoRI/SpeI) Products are Bca1015 and Bca1016 ---- ca1016F Forward EcoRI to biobrick CAT of pKD3 gacttgaattcgcggccgcttctagaggaataggaacttcatttaaatg ca1016R Reverse SpeI to biobrick CAT of pKD3 ccgctactagtggcgcgcctacctgtgacgg
The human codon optimized Cas9 from the organism Streptococcus pyogenes was standardized (RFC 25). Additionally we mutated the Nickase function. In the end we engineered a DNA binding protein, that can be fused with different effectors in order to regulate gene expression.
Restriction sites were deleted by primer overhangs. Parts were fused together by fusion PCR. The nickase function was mutated in order to abbolish the demolition of DNA.
aeBlue, blue chromoprotein (incl RBS, J23110)
aeBlue is a blue chromoprotein extracted from the basal disk of a beadlet anemone Actinia equina. The protein has an absorption maximum at 597nm and a deep blue colour. The protein aeBlue has significant sequence homologies with proteins in the GFP family.
Synthesized and codon optimized for E. coli by Bioneer Corp.
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.
Modificaitons to Clontech EYFP by Reshma Shetty
Vinay S. Mahajan, Brian Chow, Peter Carr
Generic spacer for ensuring a 70 bp distance between the end of the suffix of the BioBrick part containing the double terminator and the prefix of the BioBrick part containing the promoter of the new gene. Please, use the AlignX function of Vector NT to check for homology with the components in your plasmid before using this spacer.
The size of the spacer was choosed to meet the minimum length of a sequence that can be queried using the BLAST search engine. However, subsequences of it can be used to design shorter spacers. The sequence was selected from many more sequences randomly generated using the Random DNA Generator engine; the GC% parameter used as input was 50%. The sequences were selected based on the following constraints listed in their order of importance: the absence of any putative promoter regions, a low degree of homology with the Elowitz plasmid (whose components are widely used in our designs), no homology with other E.coli sequences as shown by BLASTN search results and the presence of a number of TAA stop codons. The second constraint was the most stringent leading to the elimination of most sequences. DE made the following changes to the original sequence in order to add stop codons in the -3 frame and more in the +2 frame (note, not all of these stop codons are UAA. Thus, if used in an organism that inserts an amino acid @ UGA or UAG the obvious will occur): T->A @ 85 T->A @ 42 C->T @ 79 A->T @ 64 A->T @ 31 T->A @ 34 C->A @ 37 Also, note that the above changes further reduce (the already very weak) homology to current NCBI-stored sequences. In the process of selecting the best sequence it appeared that a good alternative sequence for a spacer would be: AGGTTCTGATATGTAACTGTGCCCAATGTCGTTAGTGACGCATACCTCTTAAGAGGCCACTGTCCTAACA. The sequence contains no putative promoters and shows moderate homology with the 5' end of the Ampicillin resistance gene. However a strong promoter sequence starts 12 bp downstream of this sequence, and therefore the sequence presented above was preferred. The sequence is compatible (does not show significant homology) with the components in the Elowitz repressilator plasmid.
Randomly generated and optimized for several parameters (see Design notes).
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).
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. .
PDF1.2 is a plant specific promoter and 5??? untranslated region which is induced by methyl jasmonate. The part is compatible with the GoldenGate MoClo Assembly Standard as it is free from BsaI and BpiI recognition sequences.
RBS.3 (medium) -- derivative of BBa_0030
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
Weak1 RBS based on Ron Weiss thesis. Strength is considered relative to <bb_part>BBa_B0030</bb_part>, <bb_part>BBa_B0031</bb_part>, <bb_part>BBa_B0033</bb_part>.
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix ("RBS-2" in figure 4-14 of thesis). Contact info for this part: Brian Chow
PenP operator from Bacillus Lichenformis. Two operator sites are negatively regulated by PenI (C0074).
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)
MAS is a plant specific promoter which is constitutively expressed. This part is compatible with the GoldenGate MoClo Assembly Standard as it is free from BsaI and BpiI recognition sequences.
Domestication for GoldenGate by removal of BsaI and BpiI recognition sequences.
The MoClo format of BBa_J23102 with AB fusion sites.
This is the MoClo formatted part of BBa_J23102 with EB fusion sites. See the BBa_J23102 page for full information on the part. Summary of modifications from original part: Backbone has one added SpeI site in front of gene, BsaI sites, and 4bp fusion sites.
Yellow fluorescent protein (EYFP) reporter coding sequence without the Ribosome Binding Site. Modified with an LVA tail for rapid degradation of the protein and faster fall time for the emission. Annotated mutation cause a Q81L mutation that appears to not be near the active site.
BBa_E0032 yellow fluorescent protein is based on BioBrick part BBa_E0031. It has been modified to include a rapid degradation LVA tail, and includes the BioBrick standard assembly head and tail restriction sites. The RBS has been removed. The stop codon has been changed from TAA to a double stop codon TAATAA.
Modified from <bb_part>BBa_E0031</bb_part>
The MoClo format of BBa_J23106 with AB fusion sites.
This is the MoClo formatted part of BBa_J23106 with AB fusion sites. See the BBa_J23106 page for full information on the part. Summary of modifications from original part: Backbone has one added SpeI site in front of gene, BsaI sites, and 4bp fusion sites. The fusion site letters refer to 4bp fusion sites: A=GGAG; B=TACT; C=AATG; D=AGGT; E=GCTT; F = CGCT; G=TGCC; H=ACTA.
The MoClo format of BBa_J23112 with AB fusion sites.
__NOTOC__ BBa_K1114014 short BBa_K1114014 SequenceAndFeatures ===Design Notes=== This is the MoClo formatted part of BBa_J23112 with AB fusion sites. See the BBa_J23112 page for full information on the part. This is a Level 0 MoClo part with flanking sites A on the 5' side and site B on the 3' side of the part. The fusion site letters refer to 4bp fusion sites: A = GGAG; B = TACT; C = AATG; D = AGGT; E = GCTT; F = CGCT; G = TGCC; H = ACTA. Summary of modifications from original part: Backbone is a modified version of pSB1C3 with added SpeI site in front of gene, BsaI sites flanking, and 4bp fusion sites. See BBa_K783055 . ===Source=== iGEM Distribution Kit ===References===
RBS (Elowitz 1999) -- defines RBS efficiency
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix. No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info for this part: Brian Chow
amilCP blue chromoprotein with strong promoter
Blue/purple chromoprotein (amilCP: BBa_K592009) with strong promotor (CP29). The promoter is supposed to work in E. coli as well as Lactobacillus. (Construct name: pSB1C3-CP29-B0034-amilCP)
mmoC is one subunit of the mmo from Methylococcus capsulatus bath
GFP reporter for RHS of library test constructs
This part will be suffixed to a promoter.RBS library. The purpose of the experiment is to make a first pass at a library-based construction step to identify efficiency issues and also to examine maxPOPS/RIPS level.
This part was built by Jen as an intermediate to their nuts and bolts parts.
Bicistronic design RBS 12, MoClo Format with BC fusion sites
Constitutively expressed chromoprotein cjBlue
The chromoprotein cjblue is constitutively expressed and can be used for the marking of cells for easier differentiation.
This part is a combination of BBa_K1073006 and BBa_K592011, which were derived from 2013 distribution kit and the iGEM Team Uppsala.
Cyan fluorescent protein (ECFP) reporter coding sequence without the Ribosome Binding Site. Modified with an LVA tail for rapid degradation of the protein and faster fall time for the emission.
BBa_E0022 cyan fluorescent protein is based on BioBrick part BBa_E0021. It has been modified to include a rapid degradation LVA tail, and includes the BioBrick standard assembly head and tail restriction sites. The RBS has been removed. The stop codon has been changed from TAA to a double stop codon TAATAA.
Modified from <bb_part>BBa_E0021</bb_part>.
RBS-gamma protein-beta protein-exonuclease
This part is constructed for improve BBa_K1223012, pKD46 functional unit. Lambda red & pKD46 brief introduction. BBa_K1223012 is not conform to any RFC cause there are three restrict enzyme sites of EcoRI and PstI. As for improve the compatibility of this part and make these useful modules be used in future assemblies, we remove these restrict enzyme sites by synonymous mutations
Superfolder Cyan Fluorescent Protein is a derivative of superfolder Green Fluorescent protein. Compared to CFP it displays improved stability and strong cyan fluorescence.
PartsRegistry BBa_I746909, originally come from Aequorea victoria
Note: The submitted version of this part has a mutation in the BioBrick suffix: The NdeI restriction site has been lost. However, the part can still be used, because the SpeI and PstI restriction sites are functional.
Extracted from genomic DNA of ''S. cerevisiae'' using PCR.
scOrange, orange chromoprotein (incl. RBS)
Cloned from ScroogeOrange vector, DNA 2.0.
meffBlue, blue chromoprotein (incl RBS, J23110)
This chromoprotein from the coral Montipora efflorescens, meffBlue, naturally exhibits strong color when expressed.
Double terminator consisting of BBa_B0010 and BBa_B0012
Riboflavin synthesis gene cluster from shewanella oneidensis
This gene cluster consists of four different genes that form a single operon.
we eliminated three illegal restriction sites.
A CDS of Dronpa protein with a mutation in the betha-strand 7 in Lys145 for Asn.
Pectinidae sp. sequence found in the following article Lummer, M., Humpert, F., Steuwe, C., Caesar, K., Sch??ttpelz, M., Sauer, M. and Staiger, D. (2011), Reversible Photoswitchable DRONPA-s Monitors Nucleocytoplasmic Transport of an RNA-Binding Protein in Transgenic Plants. Traffic, 12: 693???702. doi: 10.1111/j.1600-0854.2011.01180.x
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.
fwYellow, yellow chromoprotein (incl RBS, J23110)
Cloned from FezziwigYFP vector, DNA 2.0.
Nitrite Reductase (nirS) from Pseudomonas aeruginosa PAO1
This gene codes for the nitrite reductase (nirS) that converts nitrite (NO2-) into nitric oxide (NO). It is cloned into the pSB1C3
The MoClo format of BBa_J23108 with AB fusion sites.
BBa_K1114011 BBa_K1114011 SequenceAndFeatures This is the MoClo formatted part of BBa_J23108 with AB fusion sites. See the BBa_J23108 page for full information on the part. This is a MoClo level 0 promoter with a flanking site A on the 5' side and site B on the 3' side of the part. The fusion site letters refer to 4bp fusion sites: A=GGAG; B=TACT; C=AATG; D=AGGT; E=GCTT; F = CGCT; G=TGCC; H=ACTA. Summary of modifications from original part: Backbone has one added SpeI site in front of gene, BsaI sites, and 4bp fusion sites.
MoClo formatted version of B0034 with BC fusion sites
This is the MoClo formatted part of BBa_B0034 with BC fusion sites. See the BBa_B0034 page for full information on the part. This is a Level 0 MoClo part with flanking sites B on the 5' side and site C on the 3' side of the part. The fusion site letters refer to 4bp fusion sites: A = GGAG; B = TACT; C = AATG; D = AGGT; E = GCTT; F = CGCT; G = TGCC; H = ACTA. Summary of modifications from original part: Backbone is a modified version of pSB1C3 with added SpeI site in front of gene, BsaI sites flanking, and 4bp fusion sites. See BBa_K783055.
The MoClo format of BBa_J23105 with AB fusion sites.
This is the MoClo formatted part of BBa_J23105 with AB fusion sites. See the BBa_J23105 page for full information on the part. Summary of modifications from original part: Backbone has one added SpeI site in front of gene, BsaI sites, and 4bp fusion sites. The fusion site letters refer to 4bp fusion sites: A=GGAG; B=TACT; C=AATG; D=AGGT; E=GCTT; F = CGCT; G=TGCC; H=ACTA.
FepA L8T Mutant - Large Diffusion pore for E. coli outer membrane.
FepA is an outer membrane receptor protein for Escherichia coli involved in the uptake of the ferric enterobactin siderophore. This part is a deletion mutant of the wild type FepA, which has been shown to convert the protein into an open diffusion channel, similar to a porin. It allows larger molecules through than normally would be permitted.
The original mutants had an EcoR1 restriction site within their coding sequence. This had to be removed via site directed mutagenesis, using a megaprimer based method.
The mutants were developed by Dr Salete Newton working in Professor P. Klebba's lab at the university of Oklahoma. They were kindly sent to us by Professor Klebba.
Jonas Demeulemeester and Benjamien Moeyaert
Fuse the LVA tag to the T7 coding region
RFC[25]-compatible dark quencher based on K1319000 (E0030)
This part is a RFC[25] dark quencher that is based upon K1319000 (the RFC[25] version of E0030/EYFP). Three mutations were introduced that eliminated fluorescence: * L90I * Y145W * H148R
Point mutations were introduced, starting from K1319000 (RFC25-YFP) as the template.
Promoter activated by RhlR in concert with C4-HSL. C4-HSL is produced by RhlI, and acts as a quorum-sensing autoinducer. This is the natural sequence taken from Pseudomonas aeruginosa. Crosstalk: this promoter is also activated at a low level by LasR with its associated HSL.
The -10 and -35 sites are labeled as identified in [Pearson97]. The part begins with the RhlR binding site, and (rather arbitrarily) ends with the first transcribed base (+1).
Pseudomonas aeruginosa rhlAB promoter
Sequence for pTet inverting regulator driven by the TetR protein.
BBa_R0040 TetR-Regulated Promoter is based on a cI promoter. It has been modified to include two TetR binding sites and the BioBrick standard assembly head and tail restriction sites.
Lutz, R., Bujard, H., Nucleic Acids Research (1997) 25, 1203-1210.
Protein generator converting TIPS to the protein cI from 434. Used as the input section for Quad Part Inverter Q01520.
Removed LVA tag from BBa_C0052 via PCR. In pSB1AK3 currently.
[LacI + LacIq promoter] reverse + [tac promoter + lac operator] forward
This is E.coli lactose-inducibile promoter. lac repressor (encoded from lacI gene) is the regulator protein: in absence of lactose the protein binds to lac operator, preventing RNA-polymerase binding and transcription. Low, constitutive levels of transcription can be seen anyhow.
EstA : inactive fusion protein for bacterial surface expression
Marie Burghard, Henrik Cordes, Jascha Diemer, Adrian Eilingsfeld, Sven Jager, Philipp Rottmann, Rene Sahm, Arne Wehling
This Esterase A is a inactivated membrane bound esterase from Pseudomonas putida. Its active site is mutated so it shows no more hydrolytic activity. It can be used for cell surface display like its shown in the following paper: Taek Ho Yang, Jae Gu Pan, Yeon Soo Seo, Joon Shick Rhee Use of Pseudomonas putida EstA as an Anchoring Motif for Display of a Periplasmic Enzyme on the Surface of Escherichia coli Appl Environ Microbiol. 2004 December; 70(12): 6968???6976. doi: 10.1128/AEM.70.12.6968-6976.2004
This certain variant of EstA should be used for cell surface expression and displaying of almost every kind of enzyme. There is no ATG at its 5' because its an C-terminal fusion protein. If you want to use you need to design it in the following way: N - Peri plasmatic signal sequence - Protein of interest - EstA - C We did not use any kind of BioBrick Assembly due to the generation of stop codons. If you would like to fuse EstA to any kind of protein, please use SOE PCR or an alternative kind of assembly strategy.
Our parts derives from an established inactive EstA mutant on pEST100 vector, which was used in the following paper: Becker, S., Theile, S., Heppeler, N., Michalczyk, A., Wentzel, A., Wilhelm, S., Jaeger, K.-E., et al. (2005). A generic system for the Escherichia coli cell-surface display of lipolytic enzymes. FEBS letters, 579(5), 1177???82. doi:10.1016/j.febslet.2004.12.087
Medium RBS based on Ron Weiss thesis. Strength considered relative to <bb_part>BBa_B0030</bb_part>, <bb_part>BBa_B0032</bb_part>, <bb_part>BBa_B0033</bb_part>.
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix ("RBS-1" in figure 4-14 of thesis). No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info for this part: Brian Cho
I746100 (AIP sensor infrastructure - agrC + agrA with RBSes) + B0015 (Terminator)
This part is simply the AIP sensor infrastructure ([http://partsregistry.org/wiki/index.php/Part:BBa_I746001 I746001]) from the agr system with a standard registry terminator ([http://partsregistry.org/wiki/index.php/Part:BBa_B0015 B0015]) downstream. The original part was made in order to transfer the S. aureus oligopeptide-based quorum sensing system into a BioBrick-compatible signalling mechanism. In the natural system, the signalling oligopeptide (termed AIP) is made from AgrD by the membrane-located enzyme AgrB. It is then detected by the membrane-located AgrC, which phosphorylates AgrA which then has DNA-binding activity and upregulates transcription of the promoters termed P2 and P3 in the agr locus. There are four known variants of AIP with different molecular structures and cross-inhibitory activity; this BioBrick generates group I AIP. This part contains the coding regions for group I agrB and agrD (in that order) from S. aureus strain NCTC8325, prefixed by the B0034 RBS and separated by a SaII site.
The original sequences for agrC and agrA were taken from the S. aureus Sequencing Project (results for strain NCTC8325) at Oklahoma University (http://www.genome.ou.edu/staph.html). The sequences were then codon-optimised for E. coli using GeneDesigner from DNA2.0 (http://www.dna20.com), and then synthesised by that company.
This chromoprotein from the coral Montipora efflorescens, meffRed, naturally exhibits color when expressed.
c2 repressor from Salmonella phage P22 (+LVA)
The P22 c2 repressor protein coding sequence is a 720 base-pair sequence with the standard RBS-compatible BioBrick prefix and the standard BioBrick suffix sections on its ends. It binds to the P22 c2 regulatory sequence, BBa_R0053. The sequence contains a LVA tag for faster degredation.
References (unparsed) here: Vander Byl,C. and Kropinski,A.M. Sequence of the genome of Salmonella bacteriophage P22 J. Bacteriol. 182 (22), 6472-6481 (2000) References (unparsed) here: Vander Byl,C. and Kropinski,A.M. Sequence of the genome of Salmonella bacteriophage P22 J. Bacteriol. 182 (22), 6472-6481 (2000)
This is the coding sequence of crtI from Pantoea ananatis (formerly Erwinia uredovora) (Accession number D90087) preceded by an E. coli synthetic ribosome binding site (rbs). It encodes phytoene dehydrogenase, part of the carotenoid biosynthesis pathway, which converts Phytoene to Lycopene.
The native crtI sequence contains two internal PstI sites. Primers crtImutf1 and crtImutf2 were ordered so that the MABEL protocol (invented for this purpose) could be used to remove these sites. The mutagenic primers were as follows: * crtImutf1: gtt tac agt aag gtt gcc * crtImutf2: agt aac tct ctg ttt gtg c In both cases, the PstI site CTGCAG was converted to CTGCAA, a silent mutation.
rbs: Escherichia coli JM109 genomic DNA. crtI: Pantoea ananatis genomic DNA
Bicistronic design RBS 13, MoClo Format with BC fusion sites
This is the MoClo formatted part of a bicistronic design ribosomal binding site 13 with BC fusion sites. This is a Level 0 MoClo part with flanking sites B on the 5' side and site C on the 3' side of the part. The fusion site letters refer to 4bp fusion sites: A = GGAG; B = TACT; C = AATG; D = AGGT; E = GCTT; F = CGCT; G = TGCC; H = ACTA. Backbone is a modified version of pSB1C3 with added SpeI site in front of gene, BsaI sites flanking, and 4bp fusion sites. See BBa_K783055.
Stephen Lee, Roshan Kumar, Joe Levine, Ziyan Chu (Polkadorks, IAP 2004)
Positively regulated, OmpR-controlled promoter. This promoter is taken from the upstream region of ompC. Phosphorylated OmpR binds to the three operator sites and activates transcription.
The promoter includes the three OmpR binding sites: C1, C2, and C3. The promoter starts at -107 and goes up to the transcriptional start site at +1. An alternate version, BBa_R0083, cuts out the C2 and C3 sites. The efficacy of this promoter versus the truncated ompC upstream region (BBa_R0083) or the ompF upstream region (BBa_R0084) is currently unknown.
contains the J23060 loop on a tRNA. more on this later
RBS.4 (weaker) -- derivative of BBa_0030
Weaker RBS based on Ron Weiss thesis. Strengths relative to <bb_part>BBa_B0030</bb_part>, <bb_part>BBa_B0031</bb_part>, <bb_part>BBa_B0032</bb_part>.
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix ("RBS-3" in figure 4-14 of thesis). No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info for this part: Brian Chow
cI repressor from E. coli phage lambda (+LVA)
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.
Thermo sensitive small RNA (ROSE structure) which can be used as a RNA regulator.
Part of the sequence is altered because of the scar...
predicted stem-loop extending to one position before start codon
EPIC Firefly Luciferase Photinus Pyralis (E. coli optimised)
PfdhF: fdhF promoter, a hypoxia inducible promoter
This tandem promoter was created using the MoClo assembly method. This is a Level 0 MoClo part with flanking sites A on the 5' side and site B on the 3' side of the part. The fusion site letters refer to 4bp fusion sites: A = GGAG; B = TACT; C = AATG; D = AGGT; E = GCTT; F = CGCT; G = TGCC; H = ACTA. The promoter is induced by atc and arabinose. The characterization data will be updated before the jamboree.
We looked through the literature and decided to attach the two promoters without any extra sequence between them
This was formed using BBa_I13453 and BBa_R0040
Positively regulated, OmpR-controlled promoter. This promoter is derived from the upstream region of ompC. Phosphorylated OmpR binds to the operator site and activates transcription. This is a truncated version of BBa_R0082.
The promoter deletes the C2 and C3 regions of BBa_R0082 (bases 37-74 in BBa_R0082) and inserts an 8-base linker before the -35 and -10 signal region. The efficacy of this promoter versus the full ompC upstream region (BBa_R0082) or the ompF upstream region (BBa_R0084) is currently unknown. The a at base 41 is mutated to t to avoid XbaI site. Speculation that this was a previous assembly site.
green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (SwissProt: P42212
GFP (mut3b) [note that this part does not have a barcode]
This is a MoClo converted version of BBa_J23114
This is a MoClo converted version of BBa_J23114. It has two MoClo fusion sites flanking it.
medium RBS, YFP (-LVA), double terminator
GldA - glycerol dehydrogenase from Escherichia coli
The Gene gldA encodes glycerol dehydrogenase from Escherichicha coli. The overexpression of glycerol dehydrogenase in E. coli can be used as a way for endogenous mediator production. Many derivates of glycerol dehydrogenase are small, water-soluble redoxmolecules, which have mediator properties. In enzymology, a glycerol dehydrogenase (EC 1.1.1.6) is an enzyme belonging to the family of oxireductases that catalyzes the chemical reaction: Glycerol + NAD --> Glycerone + NADH + H
35S is a plant specific promoter obtained from Cauliflower Mosaic Virus. The part is intended for use as a constitutive promoter for gene expression experiments. The part is compatible with GoldenGate MoClo Assembly Standard (free from BsaI and BpiI recognition sequences).
The MoClo format of BBa_J23109 with AB fusion sites.
__NOTOC__ BBa_K1114012 short BBa_K1114012 SequenceAndFeatures ===Design Notes=== This is the MoClo formatted part of BBa_J23109 with AB fusion sites. See the BBa_J23109 page for full information on the part. This is a Level 0 MoClo part with flanking sites F on the 5' side and site B on the 3' side of the part. The fusion site letters refer to 4bp fusion sites: A = GGAG; B = TACT; C = AATG; D = AGGT; E = GCTT; F = CGCT; G = TGCC; H = ACTA. Summary of modifications from original part: Backbone has one added SpeI site in front of gene, BsaI sites, and 4bp fusion sites. ===Source=== iGEM Distribution Kit ===References===
__NOTOC__ BBa_K1114033 short BBa_K1114033 SequenceAndFeatures ===Design Notes=== This is the MoClo formatted part of BBa_J23109 with FB fusion sites. See the BBa_J23109 page for full information on the part. This is a Level 0 MoClo part with flanking sites F on the 5' side and site B on the 3' side of the part. The fusion site letters refer to 4bp fusion sites: A = GGAG; B = TACT; C = AATG; D = AGGT; E = GCTT; F = CGCT; G = TGCC; H = ACTA. Summary of modifications from original part: Backbone has one added SpeI site in front of gene, BsaI sites, and 4bp fusion sites. ===Source=== iGEM Distribution Kit ===References===
amilCP, blue/purple chromoprotein (incl RBS)
This chromoprotein from the coral Acropora millepora, amilCP, naturally exhibits strong color when expressed. The protein has an absorbance maximum at 588 nm giving it a blue/purple color visible to the naked eye, thereby requiring no instruments to observe. The strong color is readily observed in both LB or agar culture, in less than 24 hours of incubation.
Illegal internal restriction site had to be removed (EcoRI).
pCooM Promoter from Rhodospirillum rubrum
The two CooA-regulated R. rubrum promoters, PcooF and PcooM, contain 2-fold symmetric DNA sequences that serve as CooA-binding sites and are similar to the CRP consensus sequence. This is consistent with the similarity between CooA and CRP in their helix-turn-helix motifs. The CooA binding sites lie at the 243.5 and 238.5 positions relative to the transcription start sites in PcooF and PcooM, respectively, overlapping with the 235 region. This overlap suggests that both CooA-regulated promoters are analogous to class II CRP-dependent promoters. We chose PcooF for this study because it is the stronger promoter based on the amount of primer extension product and level of coo-encoded proteins synthesized in vivo. The CO-dependent anaerobic growth of Rhodospirillum rubrum relies on a CO oxidation system encoded by two CO regulated transcriptional units, cooMKLXUH and cooFSCTJ. The key products of the coo regulon are an O2- sensitive CO dehydrogenase (CooS), a CooS-associated Fe-S protein (CooF), and a CO-tolerant hydrogenase (CooH), and the expression of the genes depends upon the activity of the CooA protein, which senses CO under anaerobic conditions.
Y. He, T. Gaal, R. Karls, T. Donohue, R. Gourse and G.P. Roberts, J. Biol. Chem. 274 (1999), pp. 10840???10846.
Promoter, Standard (lambda cI regulated)
Vinay S Mahajan, Brian Chow, Peter Carr, Voichita Marinescu and Alexander D. Wissner-Gross Drew Endy
[Note: This is the same part as R0051 except that the -10 and -35 sites and spacing have been changed to comply with BBa_S0001]. The cI regulated promoter is based on the pR promtoer from bacteriohage lambda. The promoter has two two DNA binding sites for lambda cI repressor <bb_part>BBa_C0051</bb_part>. cI binding results in repression of transcription. The specific sequence used here is based on the cI repressible promoter used in the Elowitz repressilator (and references therein).
In order to address concerns about the promoter transcribing in the reverse direction, we have removed the -35 and -10 signals responsible for the promoter activity in the reverse direction. (More details needed here! DE, 2/24/03) Modified to comply with BBa_S0001: TTGACA-17N-GATACT Incompatible with host expressing cI repressor.
M13 bacteriophage g8p with a FLAG tag on C-terminal
The MoClo format of BBa_J23117 with AB fusion sites.
===Design Notes=== This is the MoClo formatted part of BBa_J23117 with AB fusion sites. See the BBa_J23117 page for full information on the part. This is a Level 0 MoClo part with flanking sites A on the 5' side and site B on the 3' side of the part. The fusion site letters refer to 4bp fusion sites: A = GGAG; B = TACT; C = AATG; D = AGGT; E = GCTT; F = CGCT; G = TGCC; H = ACTA. Summary of modifications from original part: Backbone is a modified version of pSB1C3 with added SpeI site in front of gene, BsaI sites flanking, and 4bp fusion sites. See BBa_K783055 . ===Source=== iGEM Distribution Kit ===References===
Limonene synthase gene with ribosome binding site
Ribosome binding site attached to Limonene synthase gene. Limonene synthase enzyme produces (+)-limonene from farnesyl diphosphate (intermediate in mevlonate pathway). (+) Limonene is a component of lemon scent.
several codons were altered silently to remove several forbidden restriction sites. The N-terminal region (plastid targeting sequence) was removed, removing the first 51 amino acids and replacing codon 52 with the start codon ATG. LIMS cDNA was used as genomic DNA contains introns.
Monoterpene biosynthesis in lemon (Citrus limon) cDNA isolation and functional analysis of four monoterpene synthases, J Lucker, et al, Eur. J. Biochem. 269: 3160???3171 (2002) Analysis of Lemon and Bergamot Essential Oils by HPLC with Microbore Columns, M Benincasa, et al, Chromatographia 30(5/6):271 (1990) Citrus fruit flavour and aroma biosynthesis, L Sharon-Asa, The Plant Journal, 36:664 (2003)
This part is coding N-terminal torA signal region. Proteins conjugated torA signal are recognized by Tat secretion pathway and they are secreted from cytoplasm to periplasm while maintaining their holdings. This parts include RBS so that you don't bother to insert new RBS between this signal and your promoter. ( Note: Depending on your purpose, you might use BBa_K797004 when you use this signal because wild type Tat pathway is relatively saturated easily.)
When you combine two iGEM part by ligation following cutting by Xbal and Spe1, a stop codon sometimes appear. But don't worry. We modified the sequence of Tat signal so that this signal can always combine your protein.
This parts is derived from Escherichia coli???s genomic sequence.
mmoD is one subunit of the mmo from Methylococcus capsulatus bath
green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (SwissProt: P42212)
GFP-AAV [Pst1 site in earlier version knocked out]
Constitutively Expressed gRNA targeting mRFP
This Constitutively Expressed gRNA targeting mRFP is a no-BioBrick-scars assembly of the constitutive promoter BBa_J23100, DNA sequence of a gRNA that targets mRFP reporter and a reliable terminator BBa_B0015. This gRNA sequence comes from the following literature: QI, LEI S., LARSON, MATTHEW H., GILBERT, LUKE A., DOUDNA, JENNIFER A., WEISSMAN, JONATHAN S., ARKIN, ADAM P. & LIM, WENDELL A. 2013. Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression. Cell, 152, 1173-1183.
gRNA shall be exactly transcribed out. Any extra nucleotides would potentially jeopardize the normal function of the gRNA. Therefore, BioBrick scars are not allowed here. Instead of cut-and-ligation, inverse PCR or In-Fusion is preferred for the assembly task. In addition, a reliable terminator is applied here to ensure the termination of gRNA.
The gRNA sequence is de novo synthesized.
The MoClo format of BBa_I13453 with AB fusion sites.
===Design Notes=== This is the MoClo formatted part of BBa_I13453 with AB fusion sites. See the BBa_I13453 page for full information on the part. This is a Level 0 MoClo part with flanking sites A on the 5' side and site B on the 3' side of the part. The fusion site letters refer to 4bp fusion sites: A = GGAG; B = TACT; C = AATG; D = AGGT; E = GCTT; F = CGCT; G = TGCC; H = ACTA. Summary of modifications from original part: Backbone is a modified version of pSB1C3 with added SpeI site in front of gene, BsaI sites flanking, and 4bp fusion sites. See BBa_K783055 . ===Source=== iGEM Distribution Kit ===References===
Positively regulated, OmpR-controlled promoter. This promoter is taken from the upstream region of ompF. Phosphorylated OmpR binds to the three operator sites and activates transcription.
The promoter includes three OmpR binding sites: F1, F2, and F3. The promoter starts at -107 and goes up to the transcriptional start site at +1. A distant fourth binding site, F4, has been deleted. It is involved in negative regulation of ompF (Head, Tardy and Kenney, 1998), so its deletion is intended to make the promoter solely activating. The efficacy of this promoter versus the ompC upstream region (BBa_R0082) or the truncated ompC upstream region (BBa_R0083) is currently unknown.
Bicistronic design RBS 16, MoClo Format with BC fusion sites
This is the MoClo formatted part of a bicistronic design ribosomal binding site 16 with BC fusion sites. This is a Level 0 MoClo part with flanking sites B on the 5' side and site C on the 3' side of the part. The fusion site letters refer to 4bp fusion sites: A = GGAG; B = TACT; C = AATG; D = AGGT; E = GCTT; F = CGCT; G = TGCC; H = ACTA. Backbone is a modified version of pSB1C3 with added SpeI site in front of gene, BsaI sites flanking, and 4bp fusion sites. See BBa_K783055.
MoClo format of a modified version of BBa_B0032 with BC fusion sites
This is the MoClo formatted part of a modified BBa_B0032 with BC fusion sites. See the BBa_B0032 page for full information on the part. This is a Level 0 MoClo part with flanking sites B on the 5' side and site C on the 3' side of the sequence. The fusion site letters refer to 4bp fusion sites: A = GGAG; B = TACT; C = AATG; D = AGGT; E = GCTT; F = CGCT; G = TGCC; H = ACTA. This part is in a MoClo backbone that is a modified version of the pSB1C3 backbone. See <a href: http://parts.igem.org/Part:BBa_K783058???>BBA_K783058 Summary of modifications from original part: Backbone has one added SpeI site in front of gene, BsaI sites, and 4bp fusion sites.