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
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
Double terminator consisting of BBa_B0010 and BBa_B0012
crtZ (beta-carotene hydroxylase) with native rbs
Pantoea ananatis (formerly Erwinia uredovora) DSMZ 30080 (ATCC 19321). Accession: D90087.
crtZ (beta-carotene hydroxylase) from Pantoea ananatis (formerly Erwinia uredovora) DSMZ 30080 (ATCC 19321). Accession: D90087. Part of the carotenoid biosynthesis pathway. Converts beta-carotene to the yellow pigment zeaxnathin.
This part was made as a single biobrick rather than separate rbs and coding sequence, hence the use of blunt combination to generate the final sequence.
One of two requisite genes required for the biosynthesis of phycocyanobilin from heme.
phycocyanobilin:ferredoxin oxidoreductase (PcyA) from synechocystis
Second of two required phycocyanobilin biosynthetic genes.
Promoter, Standard (lambda cI regulated)
A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000
[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.
Vinay S Mahajan, Brian Chow, Peter Carr, Voichita Marinescu and Alexander D. Wissner-Gross Drew Endy
PoPS in -> lasI out. Strong RBS (efficiency=0.6). Artificial 39 bp terminator (efficiency=0.99).
This part involves the -10 binding site, the -35 binding site, and the twenty nucleotides between that constitute the lux box. With this part, LuxR functions as a acyl-homoserine lactone-dependent repressor. LuxR resonds to the HSL produced by LuxI, N-(3-oxohexanoyl)-HSL. The Lux box is positioned such that it partially overlaps the consensus -35 and -10 hexamers of an RNA polymerase binding site.
Srini Devadas, David Gray, Ronny Krashinsky, Debra Lin, and Chris Zheng Liu
CmR activated by LuxR and 3OC6HSL(AHL of Lux family)
this part express chloramphenicol resistance by the activation of LuxR and 3OC6HSL
We want to create chloramphenicol resistance gene on pSB6A1.As a vector,We already create chloramphenicol resistance gene on pSB6A1and promotor exist as biobrick(BBa_R0062).To accomplish it, vector was cutted by EcoRI and Xha while insert(promotor) was cutted by EcoRI and SpeI
Brevundimonas sp. strain SD212 (MBIC 03018)Accession: AB377271.
crtW (beta-carotene ketolase) from Brevundimonas sp. strain SD212 (MBIC 03018)Accession: AB377271. Part of the carotenoid biosynthesis pathway. Converts beta-carotene to canthaxanthin and zeaxanthin to astaxanthin.
No special considerations. Please refer to Part BBa_K395706 for more information.
alcohol acetyltransferase; converts butanol or 2-methylbutanol to butyl acetate or 2-methylbutyl ace
BBa_K395603 encodes alcohol acetyltransferase derived from MpAAT1 from Malus pumila cv. Royal Gala. MpAAT1 catalyzes the conversion of butanol or 2-methylbutanol to butyl acetate or 2-methylbutyl acetate. Butyl acetate and 2-methylbutyl acetate have a apple flavor.
RBS+lac operator+RBS+His tag+thirombin+S・tag+Factor Xa+MpAAT1+His tag+T7 terminator
Gene coding sequences come from Pantoea ananatis (formerly Erwinia uredovora) (Accession number D90087), a type of Gram negative Enterobacteriaceae. This Composite Biobrick is created by standard assembly of Part BBa_K118014, Part BBa_K118006, Part BBa_K118005 and Part BBa_K118013.
This Composite Biobrick is created by standard assembly of Part BBa_K118014 (CrtE with rbs), Part BBa_K118006 (CrtB with rbs), Part BBa_K118005 (CrtI with rbs) and Part BBa_K118013 (CrtY with rbs), which are submitted by previous iGEM teams. The whole cassette is on plasmid pSB1A2 (high copy, Ampicillin resistance). Together, enzymes CrtE, CrtB, CrtI and CrtY convert colourless farnesyl pyrophosphate to orange beta-carotene (via intermediates geranylgeranyl pyroiphosphate, phytoene and lycopene). The orange beta-carotene pigment can then be used as a coloured signal output, e.g. for biosensors. There is already individual ribosome binding site before each enzyme gene sequence. Internal restriction sites have been removed by previous iGEM teams. Please refer to Parts BBa_K118014, BBa_K118006, BBa_K118005 and BBa_K118013 for more information on individual Biobricks components. This Composite Biobrick has been put under constitutive promoter R0011 (see Part BBa_K274210) and arabinose-inducible promoter I0500 (see Part BBa_274220), transformed and tested in E.coli strain MG1655. Amount of beta-carotene produced can be measured by photospectrometer with absorbance at 455nm (beta-carotene extraction using acetone). A related Composite Biobrick is Part BBa_K274100 (CrtEBI with rbs), which "stops" at one step before beta-carotene, producing (and retaining) red lycopene pigment.
The colourless precursor, farnesyl pyrophosphate, is naturally produced in E.coli using Non-mevalonate Pathway (from pyruvate and glyceraldehyde-3-phosphate). Our experiences showed that this Biobrick works better in E.coli strains with up-regulated level of precursors. E.coli strain MG1655 demonostrates higher level of pigment (beta-carotene) production than strain TOP10.
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.
Stephen Lee, Roshan Kumar, Joe Levine, Ziyan Chu (Polkadorks, IAP 2004)
cI repressor from E. coli phage lambda (+LVA)
Elowitz, M. B. Transport, Assembly, and Dynamics in Systems of Interacting Proteins. Thesis, Princeton Univ., Princeton (1999).
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.
Vinay S Mahajan, Brian Chow, Peter Carr, Voichita Marinescu and Alexander D. Wissner-Gross
Produces LuxR protein which can sense 3OC6HSL in the media and activate transcription from R0062, the right hand Lux promoter
Promoter (lacI regulated, lambda pL hybrid)
represillator of Elowitz and Leibler (2000)
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.
Neelaksh Varshney, Grace Kenney, Daniel Shen, Samantha Sutton
Chimeric Cph1 light receptor/EnvZ protein.
Silent mutation at base 108 (G-A) to remove PstI site
lacIM1 regulated by ompC-CS1 promoter.The promoter is regulated by phosphorylated OmpR.
Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 <genbank>V01146</genbank>.
Transcription terminator for the E.coli RNA polymerase.
Suggested by Sri Kosuri and Drew Endy as a high efficiency terminator. The 5' end cutoff was placed immediately after the TAA stop codon and the 3' end cutoff was placed just prior to the RBS of Gene 1.4 (before AAGGAG). Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator.
Transcriptional terminator consisting of a 64 bp stem-loop.
Native ribosome binding site for crtZ (beta-carotene hydroxylase) from Pantoea ananatis (formerly Erwinia uredovora) DSMZ 30080 (ATCC 19321). Accession: D90087.
A virtual part to be added to the coding sequence blunt (without a scar).
crtZ (beta-carotene hydroxylase) coding sequence
Coding sequence for crtZ (beta-carotene hydroxylase) from Pantoea ananatis (formerly Erwinia uredovora) DSMZ 30080 (ATCC 19321). Accession: D90087. Part of the carotenoid biosynthesis pathway. Converts beta-carotene to the yellow pigment zeaxanthin.
RBS.1 (strong) -- modified from R. Weiss
Strong RBS based on Ron Weiss thesis. Strength is considered relative to <bb_part>BBa_B0031</bb_part>, <bb_part>BBa_B0032</bb_part>, <bb_part>BBa_B0033</bb_part>.
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix ("orig" in figure 4-14 of Ron Weiss thesis). No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info Brian Chow
Promoter (luxR & HSL regulated -- lux pR)
Promoter activated by LuxR in concert with HSL The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes.This expression is up-regulated by the action of the LuxR activator protein complexed with the autoinducer, 3-oxo-hexanoyl-HSL. Two molecules of LuxR protein form a complex with two molecules of the signalling compound homoserine lactone (HSL). This complex binds to a palindromic site on the promoter, increasing the rate of transcription.
This promoter is based on the Vibrio fischeri quorum sensing gene promoters. Two genes LuxI and LuxR and transcribed in opposite directions as shown below. The original sequence from which the parts <bb_part>BBa_R0062</bb_part> and <bb_part>BBa_R0063</bb_part> were derived is shown in the picture below. <img src="<bb_file>Image1.gif</bb_file>" width="614" height="362">
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr
strong synthetic E. coli ribosome binding site with SacI site.
This is a strong synthetic E. coli ribosome binding site. It is synthesised as two complementary oligonucleotides rather than being incorporated into a biobrick plasmid. It incorporates a SacI site overlapping the XbaI site, which is preserved when it is added to any other biobrick. This allows easy detection of the RBS after it has been added upstream of a biobrick coding sequence in a plasmid vector.
Note the presence of a SacI site overlapping the XbaI site, which is preserved when this biobrick is added to any other biobrick. At the time of writing, this biobrick is added as a short piece of DNA composed of two complementary oligonucleotides rather than being incorporated into a biobrick cloning vector by itself. It can be added upstream of a biobrick coding sequence, and its presence can easily be detected in miniprep DNA on a gel by using a SacI-SpeI or similar digest.
crtE (geranylgeranyl pyrophosphate synthase) coding sequence.
crtE (geranylgeranyl pyrophosphate synthase) from Pantoea ananatis (formerly Erwinia uredovora) DSMZ 30080 (ATCC 19321). Accession: D90087. Part of the carotenoid biosynthesis parthway.
autoinducer synthetase for PAI from Pseudomonas aeruginosa
coding region for lasI protein, which produces the chemical signal AI-1
crtB coding sequence encoding phytoene synthase
This is the coding sequence of crtB from Pantoea ananatis (formerly Erwinia uredovora) (Accession number D90087). It encodes phytoene synthase, part of the carotenoid biosynthesis pathway, which converts geranylgeranyl diphosphate to phytoene. Reference: Misawa,N., Nakagawa,M., Kobayashi,K., Yamano,S., Izawa,Y., Nakamura,K. and Harashima,K. Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products expressed in Escherichia coli. J. Bacteriol. 172 (12), 6704-6712 (1990)
modified E. coli thr terminator, replaced all A-T pairs in stem with C-G pairs
Artificial terminator, estimated %T~>90% *8bp stem, 6nt loop *Bidirectional, estimated reverse %T~>90%
Bidirectional, with the reverse estimated to be less effective than the forward. Has a polyA tail of 9 residues.
Synthetic ribosome binding site added by Son of Babel procedure
This is a 4bp synthetic ribosome binding site which can be added by fusion PCR using a reverse primer and the Son of Babel cloning system.
The part can be added by fusion PCR using a Son of Babel reverse primer.
aaaatatatcatca tgaacaataaaactgtctgcttacataaacagtaatacaaggggtgttatgagccatattcaacgggaaacgtcttgctcccgtccgcgcttaaactcca acatggacgctgatttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgcttgtatgggaagcccgatgcgccaga gttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtccgtctcaactggctgacggagtttatgcctctcccgaccatc aagcattttatccgtactcctgatgatgcgtggttactcaccaccgcgattcctgggaaaacagccttccaggtattagaagaatatcctgattcaggtg aaaatattgttgatgcgctggccgtgttcctgcgccggttacattcgattcctgtttgtaattgtccttttaacagcgatcgtgtatttcgtcttgctca ggcgcaatcacgcatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcacaag ctcttgccattctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaattaataggttgtattgatgttg gacgggtcggaatcgcagaccgttaccaggaccttgccattctttggaactgcctcggtgagttttctccttcattacagaaacggctttttcaaaaata tggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttctaataa
We get this part ,RBS-CmR, from BBa_P1004 by PCR. this doesn't have any promoters and terminators.
We want to create promoterless CmR on pSB6A1. so We get this part ,RBS-CmR, from BBa_P1004 by PCR and then ligated with pSB6A1
crtW (beta-carotene ketolase) coding sequence
Coding sequence for crtW (beta-carotene ketolase) from Brevundimonas sp. strain SD212 (MBIC 03018)Accession: AB377271. Part of the carotenoid biosynthesis pathway. Converts beta-carotene to canthaxanthin and zeaxanthin to astaxanthin.
ATGA TGTCCTTTTC CGTACTGCAA GTGAAACGTC TGCAACCGGA 301 ACTGATTACA CCGGCAAAAT CAACCCCTCA GGAAACCAAA TTTCTGAGCG ACATCGATGA 361 TCAAGAATCT CTGCGCGTTC AGATTCCGAT TATCATGTGT TATAAAGACA ACCCGAGCCT 421 GAATAAAAAC CGTAACCCGG TCAAAGCCAT TCGTGAAGCA CTGTCACGTG CCCTGGTCTA 481 TTATTATCCA CTGGCCGGTC GTCTGCGTGA AGGTCCAAAC CGTAAACTGG TGGTCGATTG 541 TAACGGCGAA GGCATCCTGT TTGTGGAAGC TTCCGCCGAT GTTACGCTGG AACAGCTGGG 601 GGACAAAATC CTGCCTCCTT GTCCTCTGCT GGAGGAGTTT CTGTATAACT TCCCGGGTTC 661 TGATGGCATT ATCGACTGCC CACTGCTGCT GATTCAAGTG ACCTGTCTGA CTTGTGGAGG 721 GTTCATCCTG GCTCTGCGCC TGAACCATAC AATGTGTGAT GCCGCCGGTC TGCTGCTGTT 781 TCTGACCGCT ATTGCCGAAA TGGCCCGTGG CGCTCATGCC CCTAGTATTC TGCCTGTCTG 841 GGAGCGTGAA CTGCTGTTTG CTCGTGATCC TCCTCGTATC ACCTGTGCCC ACCATGAATA 901 TGAGGACGTG ATTGGCCACT CAGATGGGTC TTATGCCAGC TCAAATCAGA GTAATATGGT 961 CCAACGCTCG TTCTATTTTG GCGCCAAAGA GATGCGCGTA CTGCGTAAAC AAATTCCGCC 1021 ACACCTGATT AGCACTTGTA GCACCTTCGA TCTGATCACC GCTTGTCTGT GGAAATGTCG 1081 TACCCTGGCA CTGAACATTA ACCCGAAAGA GGCCGTTCGT GTTTCGTGTA TTGTGAATGC 1141 TCGTGGTAAA CACAACAACG TTCGCCTGCC TCTGGGTTAT TATGGTAACG CCTTTGCCTT 1201 TCCGGCAGCA ATTTCAAAAG CCGAACCTCT GTGTAAAAAC CCGCTGGGAT ATGCTCTGGA 1261 ACTGGTAAAA AAAGCGAAAG CGACTATGAA CGAAGAGTAT CTGCGTTCTG TTGCTGATCT 1321 GCTGGTACTG CGCGGTCGCC CTCAGTATAG TAGTACCGGC TCCTATCTGA TCGTTAGCGA 1381 CAACACCCGT GTAGGCTTCG GAGATGTGAA TTTTGGCTGG GGTCAGCCTG TTTTTGCTGG 1441 ACCGGTTAAA GCACTGGATC TGATCTCGTT TTATGTCCAA CACAAAAACA ACACCGAGGA 1501 TGGCATTCTG GTTCCGATGT GTCTGCCTAG TAGCGCTATG GAACGCTTTC AGCAGGAACT 1561 GGAGCGTATT ACCCAGGAAC CAAAAGAGGA CATCTGTAAC AATCTGCGCT CAACGTCCCA 1621 GTAA
V. fischeri <genbank>AF170104</genbank>
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.
crtI coding sequence encoding phytoene dehydrogenase
This is the coding sequence of crtI from Pantoea ananatis (formerly Erwinia uredovora) (Accession number D90087). 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.
crtY coding sequence encoding lycopene B-cyclase
This is the coding sequence of crtY from Pantoea ananatis (formerly Erwinia uredovora) (Accession number D90087). It encodes lycopene B-cyclase, part of the carotenoid biosynthesis pathway, which converts lycopene to B-carotene.
Lutz, R., Bujard, H., Nucleic Acids Research (1997) 25, 1203-1210.
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.
June Rhee, Connie Tao, Ty Thomson, Louis Waldman
tetracycline repressor from transposon Tn10 (+LVA)
Elowitz, M. B. Transport, Assembly, and Dynamics in Systems of Interacting Proteins. Thesis, Princeton Univ., Princeton (1999)
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.
June Rhee, Connie Tao, Ty Thomson, Louis Waldman.
PompC(CS1) Promoter regulated by phosphorylated OmpR
Conversion of the iGEM parts registry to SBOL2.1