Promoter, Standard (HK022 cI regulated)
[Note: This is the same part as R0050 except that the -10 and -35 sites and spacing have been changed to comply with BBa_S0001]. The pR regulatory region is a 98 base-pair sequence with standard BioBrick prefix and suffix sections on its ends. It contains two operator sites at which HK022 cI protein can bind cooperatively. The -35 and -10 regions from pR are included along with some flanking sequences. The cutoff for the 5' end of the regulatory region was placed just after the pM -10 region which promotes transcription of a gene upstream (thus the pM -10 region is not included). The cutoff for the 3' end of the regulatory region was placed immediately prior to the RBS.
Derived from STHK022N . Modified to comply with BBa_S0001: TTGACA-17N-GATACT Literature claims that there is no cross-talk between HK022 cI regulatory region, Lambda cI, 434 cI and P22 cI, but this has not been verified experimentally by the synthetic biology group at MIT.
One of two requisite genes required for the biosynthesis of phycocyanobilin from heme.
cI is a repressor of the phage lamda. It binds specificly to a operator site ( BBa_R0051 & BBa_K105021).
To enable the fusion with other domains in frame the vector of this BioBrick has no base pair in between the restriction side and the BioBrick. Furthermore, this coding sequence does not include a start codon.<br\n> For more information about this issus, see:<br\n> Phillips, I.E. and Silver, P.A. "A new Biobrick Assembly Strategy Designed for Facile Protein Engineering." <br\n> DSpace http://hdl.handle.net/1721.1/32535 (2006).
We changed the format of the cI coding part of BBa_C0051 into Phillips/Silver standard.
This part is induced by AI-2 to produce YFP.
This part accepts POPS to generate EYFP under the medium strength RBS B0032.
Barry Canton [bcanton@mit.edu] and Anna Labno [labnoa@mit.edu]
This device senses the 3OC6HSL concentration in the media and produces output PoPS according to the transfer function given below. LuxR production is controlled by the tet repressible R0040. Initial characterization work has been done on this device using I13273 as a test device.
Example Data Sheets for F2620
The Lac Y gene has been PCR amplified from the chromosomal DNA of MG1655 (Escherechia coli)and has been inserted into plasmid pSB1A2( Part number BBa_B0015).
rhlR repressor/activator from P. aeruginosa PA3477 (no LVA)
mCherry, bacterial with RBS and forward terminator
Combines BBa_B0015 forward terminator with BBa_J06602 mCherry, bacterial with RBS
green fluorescent protein derived from A. victoria GFP (SwissProt: P42212) with PEST191 tag
GFP-linker-PEST191 is a part composed of GFP (as a reporter) linked with PEST191 through six aminoacid long protein linker. PEST191 is a sequence that leads towards degradation of protein linked with this sequence. It's estimated half-time is 2h.
GFP-linker-PEST191 is made by PCR overlap extension technique.
This is the a constitutive GFP reporter driven by the cI promoter.
This gene encodes the N-terminal 77 amino acid residues of LacZ. When expressed in an E. coli host carrying the lacZ-delta-M15 mutation, common in laboratory strains, it complements the delection resulting in the production of active LacZ, which can be detected by various means, including chromogenic substrates such as Xgal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside) and ONPG (o-nitrophenyl galactoside).
Note that a stop codon was introduced to replace codon 78 of lacZ, resulting in production only of the N-terminal 77 amino acid residues of LacZ. This is sufficient to complement the lacZ-delta-M15 mutation commonly found in laboratory strains of E. col used for alpha-complementation.
The DNA was derived by PCR from E. coli BL21, which possesses an intact lacZ gene. Primers were designed based on sequence from GenBank accession J01636, GI:146575. A stop codon was artificially introduced to replace codon 78. The sequence reported here is derived by sequencing of the Biobrick construct.
tRNA_Arg5 to use as transcriptional reporter
tRNA_Arg5 to use as transcriptional reporter. Sequence is natural tRNA_Arg5 except with A to U mutation near its 3' end.
TetR promoted AHL sensor and LacI producer
This part produces LuxR when no tetR is present, which forms a complex with AHL to activate pLuxR promotor leading to LacI transcription.
tccctatcagtgatagagattgacatccctatcagtgatagagatactgagcactactagagaaagaggagaaatactagatgaaaaacataaatgccgacgacacatacagaataattaataaaattaaagcttgtagaagcaataatgatattaatcaatgcttatctgatatgactaaaatggtacattgtgaatattatttactcgcgatcatttatcctcattctatggttaaatctgatatttcaatcctagataattaccctaaaaaatggaggcaatattatgatgacgctaatttaataaaatatgatcctatagtagattattctaactccaatcattcaccaattaattggaatatatttgaaaacaatgctgtaaataaaaaatctccaaatgtaattaaagaagcgaaaacatcaggtcttatcactgggtttagtttccctattcatacggctaacaatggcttcggaatgcttagttttgcacattcagaaaaagacaactatatagatagtttatttttacatgcgtgtatgaacataccattaattgttccttctctagttgataattatcgaaaaataaatatagcaaataataaatcaaacaacgatttaaccaaaagagaaaaagaatgtttagcgtgggcatgcgaaggaaaaagctcttgggatatttcaaaaatattaggttgcagtgagcgtactgtcactttccatttaaccaatgcgcaaatgaaactcaatacaacaaaccgctgccaaagtatttctaaagcaattttaacaggagcaattgattgcccatactttaaaaattaataacactgatagtgctagtgtagatcactactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttatatactagagacctgtaggatcgtacaggtttacgcaagaaaatggtttgttatagtcgaataaatcacacaggactactagatggtgaatgtgaaaccagtaacgttatacgatgtcgcagagtatgccggtgtctcttatcagaccgtttcccgcgtggtgaaccaggccagccacgtttctgcgaaaacgcgggaaaaagtggaagcggcgatggcggagctgaattacattcccaaccgcgtggcacaacaactggcgggcaaacagtcgttgctgattggcgttgccacctccagtctggccctgcacgcgccgtcgcaaattgtcgcggcgattaaatctcgcgccgatcaactgggtgccagcgtggtggtgtcgatggtagaacgaagcggcgtcgaagcctgtaaagcggcggtgcacaatcttctcgcgcaacgcgtcagtgggctgatcattaactatccgctggatgaccaggatgccattgctgtggaagctgcctgcactaatgttccggcgttatttcttgatgtctctgaccagacacccatcaacagtattattttctcccatgaagacggtacgcgactgggcgtggagcatctggtcgcattgggtcaccagcaaatcgcgctgttagcgggcccattaagttctgtctcggcgcgtctgcgtctggctggctggcataaatatctcactcgcaatcaaattcagccgatagcggaacgggaaggcgactggagtgccatgtccggttttcaacaaaccatgcaaatgctgaatgagggcatcgttcccactgcgatgctggttgccaacgatcagatggcgctgggcgcaatgcgcgccattaccgagtccgggctgcgcgttggtgcggatatctcggtagtgggatacgacgataccgaagacagctcatgttatatcccgccgttaaccaccatcaaacaggattttcgcctgctggggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcactggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagc gggcaggctgcaaacgacgaaaactacgctttagtagcttaataactctgatagtgctagtgtagatctctactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttata
This is one of the constructs used to characterise superfolder GFP (see I746916 part description for source and other information about this GFP variant)
This part was created by standard assembly from construction intermediates: I746911 was put together with I746916:B0015
Artificial terminator, estimated %T~=70% *8bp stem, 6nt loop *Bidirectional, estimated reverse %T~=80%
Bidirectional, with the reverse estimated to be more effective than the forward. Has a polyA tail of 5 residues.
modified E. coli thr terminator, replaced all A-T pairs in stem with C-G pair
constitutive promoter family member and RBS
Constitutive promoter (strong) with RBS (strong, efficiency=0.3)
Promoter: John Anderson. RBS: Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
Mutation has been sequenced. The rest of the part will be sequenced shortly.
Part for screening plasmid test (to be combined with an upstream inverter library).
This is a high level constitutive promoter from the yeast gene TEF2.
From the yeast genome using promoter definition from: Mumberg, Muller, and Funk: http://dx.doi.org/10.1016/0378-1119(95)00037-7
Inverting regulatory region controlled by LacI (<bb_part>BBa_C0010</bb_part>, <bb_part>BBa_C0011</bb_part>, etc.) The pLac regulatory region is a 243 base-pair sequence with standard BioBrick prefix and suffix sections on its ends. It contains two protein binding sites: CAP, which is generally present in E.coli and is assocciated with cell health and availability of glucose., and LacI, the Lac inhibitor <bb_part>BBa_C0010</bb_part> which binds in an dimerized cooperative manner to inhibit the transcription of the protein that follows. In the presence of lactose or IPTG, an analog of lactose, LacI is unable to correctly bind and inhibit transcription. This allows <bb_part>BBa_R0010</bb_part> to be used as a inverter or as a detector of lactose or IPTG.
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 this regulator. This part is incompatible with environments containing lactose or lactose analogs.
The Plac insert was PCR'd from the MG1655 strain of E.coli K12.
BBa_J04700 (ForAptRibo) Riboswitch with BBa_J04671 (EYFP, RBS-, LVA+, B0015)
Andrew Drysdale and Kristen DeCelle (also Oscar Hernandez and Matt Gemberling)
This is a (ForAptRibo) Riboswitch device that contains a EYFP reporter region with an LVA tag and a B0015 Terminator Sequence
SV40 nuclear localization sequence from SV40; yeast codon optimized
yeast codon-optimized nuclear localization sequence from simian virus 40
direct synthesis from published sequence
This construct is used to screen the level of induction of the P2 ogr activator on the PF promoter. Note that the I746350 does contain a RBS.
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 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.
Tc sensor and cl lambda generator (Tet promoter)
This part serves as a sensor and reporter of intracellular tetracycline concentrations as well as producing cl (lambda) corrosponding to the level of Tc. This part uses the tet promoter to drive production of TetR. The sensor and reporter portions are identical to Part:BBa_I744104.
The promoter driving tetR production is crucial to the sensor's sensitivity and range of detection.
Composite - all parts are drawn from the registry
excisionase from E. coli phage lambda (removes prophage from host genome)
Lambda excisionase coding region with AAV degradation tag and two stop codons
This device is a PoPS in -> lasI out protein generator with Plas promoter downstream. It is a module of the following genetic multiplexer (mux): {|align="center" | |- | |} where: *PA, PS and PB are three promoters that can be ACTIVATED by the three generic elements A, S and B respectively; *GOI is the ''gene of interest'' that is the output of the system. In particular, '''K081001 is Channel 1'''. People who want to use the genetic mux have to assemble: *K081000 under the desired regulation for Channel 0 (for example the generic promoter PA); *K081001 under the desired regulation for Channel 1 (for example the generic promoter PB); *K081002 under the desired regulation for Selector (for example the generic promoter PS); *the desired protein generator downstream of K081000 and K081001 (for example the generic RBS-GOI-T generator);
mRFP (DsRed) derived, Ex587/Em610, yeast codon optimized
Codon optimized for yeast. Contains N- and C-terminal linker sequences (with homology to biobrick parts BBa_E2050 (mOrange), BBa_E2020 (Cerulean CFP), and BBa_E2030 (Venus YFP) to facilitate color swapping in yeast. Adds N-"MATSG" and "GSGTA"-C. to published amino acid sequence. Double TAATAA stop codon. Missing EcoRI, HindIII, NotI, NdeI, XhoI, RsrII, BamHI, NcoI, BglI, SpeI, XbaI, and PstI. except for 5' and 3' ends no significant sequence identity runs to mOrange (BBa_E2050).
Shaner et al, 2004. Nat Biotech (22):1567-1571
RBS.1 (strong) -- modified from R. Weiss
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
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
luxR protein generator under Plambda regulation
This device produces luxR constitutively under the strong promoter Plambda, in absence of cI repressor. Plambda can be turned off by cI repressor and luxR production cannot continue. Strong RBS (efficiency=0.6). Artificial 39 bp terminator (efficiency=0.99).
Lux Receiver (I13263 with reversed part order)
Receiver circuit similar to I13263 but with the order of the reporter and luxR generator reversed
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
PoPS in -> lasI out. Strong RBS (efficiency=0.6). Artificial 39 bp terminator (efficiency=0.99).
This is a reverse version of BBa_C0012, the lacIq repressor cds.
Standard biobrick prefix and suffix were used. Since PCR was used, DNA sequence verification was needed - no mutations were found.
PCR was used to amplify C0012 using primers that switched the biobrick prefix and suffix.
luciferase: luciferin 2-monooxygenase from Renilla reniformis (EC 1.13.12.5; SwissProt:: P27652)
Rluc is a protein called Renilla`s luciferase and it emits light when adding the right substrate. That is why it can bi used as a reported to track other proteins or to monitor the activity of a promotor.
Rluc is cloned in BioBrick vector pSB1AK3.
intermediate for lac ring oscillator synchronization device
Ring Oscillator Synchronization Device (lac)
This device is meant to be coupled to the elowitz repressilator device to synchronize a population of oscillating cells.
Lars Velten, Simon Haas, Hannah Meyer, Anne Rademacher, Hannah Uckelmann and Corinna Hiller
This device is a PoPS in -> luxI out protein generator with Plux promoter downstream. It is a module of the following genetic multiplexer (mux): {|align="center" | |- | |} where: *PA, PS and PB are three promoters that can be ACTIVATED by the three generic elements A, S and B respectively; *GOI is the ''gene of interest'' that is the output of the system. In particular, '''K081000 is Channel 0'''. People who want to use the genetic mux have to assemble: *K081000 under the desired regulation for Channel 0 (for example the generic promoter PA); *K081001 under the desired regulation for Channel 1 (for example the generic promoter PB); *K081002 under the desired regulation for Selector (for example the generic promoter PS); *the desired protein generator downstream of K081000 and K081001 (for example the generic RBS-GOI-T generator);
T7rnap (T7 RNA Polymerase) - ATG Start Codon
This is the T7 RNA Polymerase gene's Open Reading Frame, and it will then transcribe any genes with a T7 Promoter, when this polymerase is produced. It provides very good PoPS (Polymerases Per Second) output, as well as it is highly orthogonal to E. Coli's natural transcription system.
Untagged GFP behind a constitutive promoter.
[TetR][rbs][LuxR][dblTerm][LuxPR]+[rbs][LacI][dblTerm] "AHL-dependent inverter"
part of a NAND-gate system with parts ____
This part is used to screen the level of induction of the Psid promoter (I746364). The Psid promoter is induced by a class of phage activators (P2 ogr: I746350, PSP3pag: I746351, phiR73 delta: I746352).
The Psid promoter was put upstream of I13504.
registry (I13504) and own construct (I746364)
This is composed of a promoter and a GFP reporter gene. This can produce green fluorescence and in theory the strength of the green flourence is much stronger when there is phosphorylated AgrA in the cell. This is used to test the basal activity of agr P2 promoter.
We used two subparts from the registry to produce the new part. BBa_I746104+BBa_E0840
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)
Reporter Construct (Ly-Tc-) for constitutive CFP and inducible YFP
Chris Batton, Victoria Chou, Kenneth Nesmith
Reporter plasmid with Lac operator+YFP(LVA-) reporter followed by a Tet operator+CFP(LVA-) reporter
This device is a PoPS in -> pcyA out with Pomp promoter downstream. Pomp (BBa_R0082) is a positively regulated, OmpR-controlled promoter. Phosphorylated OmpR binds to the three operator sites and activates transcription. Proteins pcyA, ho1 and cph8 can produce a photosensitive complex that can activate the endogenous transcription factor OmpR. So, light (in particular 660 nm radiation) is an indirect repressor of Pomp. These interactions are valid if used in E.coli deficient in natural EnvZ, because EnvZ can activate OmpR. {|align="center" |}
Intermediate in two promoter screening plasmid
Tet operator with CFP reporter (with LVA tag) [R/Tc+]
Christopher Batten, Victoria Chou, Kenneth Nesmith
enhanced yellow fluorescent protein derived from A. victoria GFP
Artifical terminator, estimated %T~=80 6bp stem, 4nt loop Bidirectional, estimated reverse %T=40
Bidirectional, with the reverse estimated to be less effective than the forward. Has a polyA tail of 5 residues.
It is an imortant protein in signaling cascade through TLr receptors (MyD88 dependent pathway).
Protein generator converting TIPS to the protein cI from lambda. Used as the input section for Quad Part Inverters Q01150 and Q01151.
Strong RBS (efficiency=0.6) - cI repressor - Strong RBS (efficiency=0.6).
This is meant to serve as a reporter device for the synchronized oscillator.
lasI: autoinducer synthetase for PAI from Pseudomonas aeruginosa. Strong RBS (efficiency=0.6).
The 434 cI regulatory region sequence is a 89 base-pair sequence with the standard BioBrick prefix and suffix sections on its ends. 434 cI repressor protein, <bb_part>BBa_C0052<bb_part>, binds to it. This segment contains O-R1, O-R2, P-R, and P-RM -35.
Overlap extension of synthetic oligonucleotides
Reverse Complement of P22 attB site (B'-O-B)
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.
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.
pBAD is an E.coli promoter that is induced by L-arabinose. K206000 is a mutagenized pBAD promoter that is responsive to lower concentrations of arabinose than wild type ( I13453 ) and, additionally, exhibits a higher maximum of protein expression as measured by coupling to a fluorescent reporter.
There were no special design considerations.
The sequence was obtained by applying all mutations that upregulated AraC binding and subsequent promoter activity listed in reference [1].
5??? Integration Sequence for the amyE locus of B. subtilis
The 5' integration sequence can be added to the front of a Biobrick construct and the 3' integration sequence specific for this locus (Part BBa_K143002) to the rear of the Biobrick construct to allow integration of the Biobrick construct into the chromosome of the gram positive bacterium B.subtilis. The AmyE locus was the first locus used for integration into ''B.subtilis'' by Shimotsu and Henner#1 and is still commonly used in vectors such as pDR111#2, pDL#3 and their derivatives. Integration at the AmyE locus removes the ability of ''B.subtilis'' to break down starch, which can be assayed with iodine as described by Cutting and Vander-horn#4. The 5' and 3' integration sequences for the AmyE locus were used to integrate the Imperial 2008 iGEM project primary construct into the ''B.sutbilis'' chromosome. #1 pmid=3019840 #2 pmid=14597697 #3 ''Bacillus'' Genetic Stock Center [www.bgsc.org] #4 Cutting, S M.; Vander-Horn, P B. Genetic analysis. In: Harwood C R, Cutting S M. , editors. Molecular biological methods for Bacillus. Chichester, England: John Wiley & Sons, Ltd.; 1990. pp. 27???74.
The AmyE integration sequence was taken from the vector after comparison by BLAST to the ''B.subtilis'' chromosome to identify the homologous sequences. The sequence present in both the host chromosome and the plasmid at the 5' end of the gene is the 5' sequence required for integration.
The 5??? integration sequence was taken from the shuttle vector pDR111 which has been used in many studies on ''B.subtilis'', in particular in the studies of transcriptional control#1 #2 #3 #1 pmid=14597697 #2 pmid=15937167 #3 pmid=12169614
COMT (caffeic acid-O-methyl transferase) with ribosome binding site. Enzyme methylates -OH on C4 of the caffeic acid aromatic ring to produce ferulic acid. COMT is the third gene in the vanillin biosynthesis pathway,
Used COMT cDNA as genomic DNA contains introns
Genes and Enzymes Involved in Caffeic Acid Biosynthesis in the Actinomycete Saccharothrix esanaensis, Berner M, et al, Journal of Bacteriology 188(7): 2666, (2006) Gowri G, et al, Molecular cloning and expression of alfalfa S-adenosyl-L-methionine: caffeic acid 3-0-methyltransferase, a key enzyme of lignin biosynthesis, Plant Physiology 97(1):7 (1991) Downregulation of Caffeic Acid 3-O-Methyltransferase and Caffeoyl CoA 3-O-Methyltransferase in Transgenic Alfalfa Impacts on Lignin Structure and Implications for the Biosynthesis of G and S Lignin, Guo D, et al, Plant Cell. January; 13(1): 73 (2001)
rbs+appY (encoding a DNA-binding transcriptional activator)
This is the coding sequence of appY from Escherichia coli JM109 preceded by a synthetic ribosome binding site (rbs). It encodes a transcriptional regulator related to anaerobic energy metabolism. Overexpression of the gene has been reported to result in enhanced lycopene production by E. coli (Kang et al., 2005).
This construct is used to screen the level of induction of the phiR73 delta activator on the PP promoter. Note that the I746352 does contain a RBS.
The third screening plasmid. Same as the second except with the flurophores switched. The ends should be capped with I13450.
This device can produce a lactonase. Normally, however, de production of lactonase is inhibited by the lock. When key is added (or produced) the production of lactonase will occur.
This part accepts POPS to produce a single mRNA encoding ECFP and EYFP under the medium RBS B0032
mRFP (DsRed) derived, altered to be a biobrick by removing a PstI site and adding in the ends.
Made a point mutation to eliminate a PstI site in the middle and then added BioBrick ends with the LVA tag using PCR. Sequenced using primers that bind to the pSB1A2 plasmid on either side of the brick.
mCherry from Roger Y. Tsien's lab, altered to be BioBrick compatible and with an LVA tag added.
'dif' (division induced filamentation) is the recombination site for XerC and XerD recombinases. Once replicated, aligned direct dif repeats are vital to DNA monomerisation during bacterial septation. As with other recombination sites, inverted repeats cause inversion of the DNA in between. However, XerCD requires activation by FtsK and thus two dif-sites (see also part BBa_I742102) enable recombination that is temporally isolated to division.
PabA is one of three genes (pabA, pabB, pabC) involved in the para-aminobenzoic acid (pABA) synthesis pathway from chorismate. In this construct, pabA (para-aminobenzoate synthetase component II) has been placed behind the strong promoter j23100 and the strong RBS b0034, and in front of the terminator b0015. Currently it is in the inducible copy plasmid psb2k3, which can be induced to high copy via IPTG.
The purpose of this construct is to test the effects of pabA overexpression on the total production of pABA from chorismate. pABA is a necessary structural component in the synthesis of folate and so the hope is that by increasing the flux through the pABA synthesis pathway, we will be able to increase the total production of folate.
pabA has been extracted from the genome of L.lactis subspe. IL1403.
Composite part based on the A340620 PoPS amplifier. pLuxL promoter driving luxR-based HSL receiver. pLux-R is driving a YFP output device.
Built by Josh as an intermediate in screening plasmid construction
This part consists of the cstA promoter, which is repressed by high levels of glucose, coupled to the lacZ' reporter gene for promoter activity assays.
The cstA promoter and lacZ' are both from Escherichia coli.
This construct is used to screen the level of induction of the PSP3 pag activator on the PLL promoter. Note that the I746351 does contain a RBS.
Promoter, Zif23 regulated, test: between
Promoter with standard -10 and -35 sites. A symmetric Zif23 dimer target site is between the -10 and -35 sites.
consensus sequences based on [Jensen98].
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.
modified E. coli thr terminator, replaced all A-T pairs in stem with C-G pairs
This part accepts POPS to produce a single mRNA encoding ECFP and EYFP under weak RBS B0031
BglII and BamHI restriction sites with some flanking
http://openwetware.org/wiki/Silver:_Oligonucleotide_Inserts Oligonucleotide Inserts
This construct is used to screen the level of induction of the PSP3 pag activator on the PP promoter. Note that the I746351 does contain a RBS.
PoPS in -> GFP out Strong RBS (efficiency=0.6).
The first screening plasmid. Ends will be capped with I13450. Cutting I13452 generates an internal BioBrick site.
Adds an RBS to LacI protein coding region
PC and AraC from the AraBAD operator. PC and AraC are on the complementary strand, reading right to left.
Promoter, Zif23 regulated, test: between and after
Promoter with standard -10 and -35 sites. A symmetric Zif23 dimer target site is between the -10 and -35 sites, and a symmetric Zif23 dimer target site is after the -10 site.
Vikram Vijayan, Allen Hsu, Lawrence Fomundam
LasR and 3OC12HSL regulated promoter with a single Tet Operator (binding site for TetR repressor)
Zif23-GCN4 engineered repressor (+LVA, C2000 codon-optimized for E.coli)
Variant of Zif23-GCN4 (see BBa_C2000). CDS has been optimized (via VNTI) for expression in enteric bacteria. ssrA tag (LVA) added.
This protein should form a dimer and bind a specific operator site, thus repressing transcription.
Protein Engineering Gods from the Pabo Lab
Modified lamdba Prm promoter (cooperative repression by 434 cI)
Lamdba Prm promoter modified to be activated by lamda repressor (cI) and repressed by 434 repressor (cI). Two operators are used for both activation and repression to enhance cooperativity.
The O-R3 site of lambda (17 nt) was replaced by the O-R1 site of 434 (14 nt) to facilitate repression by 434 cI. The new 434 O-R1 is 5'-justified with the lambda O-R3 it replaced, leaving the -10 sequence intact. The 434 O-R2 site replaces the wildtype labmda promoter starting at -5, thereby preserving the 8 nt between 434 O-R1 and O-R2 in the wildtype. The mRNA transcript will contain an additional 9 nt compared to wildtype lambda phage.
Bushman, F. D. The bacteriophage 434 right operator roles of O-R1, O-R2, and O-R3. J. Mol. Biol. (1993) 230, 28-40.
The Isaacs key1 riboregulator taR12 is modeled on the hok/sok post-segregational killing method of plasmid R1. It makes 25 base pairs with its counterpart, the riboregulator J01010 (aka "lock 1" or "crR12"). Isaacs documentation (see reference #1): Upon induction of the araC promoter (using 0.25% arabinose solution), the taR12 key activated the corresponding crR12 lock to express the downstream indicator, GFP, to levels of fluorescence 19-fold greater than without taR12 expression. Maximal expression was reached at 70 minutes after addition of arabinose solution, and was maintained after that point.
Double terminator with two copies of <bb_part>BBa_B0010</bb_part>
GFP Producer Controlled by 3OC6HSL Receiver Device
The luxR based receiver, F2620 (formerly I13270), controls the production of GFP. The GFP protein generator is the same as that found in I7101.
Reverse RBS (RBSrev) -- corresponds to BBa_B0030
Repeats in oligos caused unusual products during cloning
chosen a medium-weak constitutive promoter. In order to have strong repression but also easy derepression by aTc.
Promoter can be inserted into the adapter containing BglII and BamHI sites. The activity of inserted promoter can be quantificationally measured using ONPG assay or fluorescent assay.
protein A from Staphylococcus aureus. Also known as part of Tap-Tag
This part is an intermediate to the protein generator BBa_45400.
This construct is used to screen the level of induction of the P2 ogr activator on the PO promoter.
Right facing promoter (for xylF) controlled by xylR and CRP-cAMP
potential looping feature possibly removed
removed RBS, left promoter, and several xylR binding sites
'dif' (division induced filamentation) is the recombination site for XerC and XerD recombinases. Once replicated, aligned direct repeats of the dif site are vital to DNA monomerisation during bacterial septation. As with other recombinase sites, inverted repeats cause inversion of the DNA in between. However, XerCD requires activation by FtsK and thus two dif-sites (see also part BBa_I742102) enables recombination that is temporally isolated to septation.
Constructed as two complimentary oligonucleotides.
rhlR repressor/activator from P. aeruginosa PA3477 (+LVA)
Debra Lin, Srini Devadas, David Gray, Ronny Krashinsky, and Chris Zheng Liu, Boopers
Transcriptional regulator, in complex with N-butyryl-HSL, RhlR binds to the Rhl promoter
(1/28/04 Edit Jamboree) (0) No BB sites (1) ATG (2) TAATAA (3) Blast checks out (4) No codon changes (5) Maybe chassis compatability (check by experiment) (6) Codon usage not optimal (but looks OK) (7) ssrA tag added
RcsB is a receiver protein in a two component phosphorelay system. Up-regulation of RcsB has been shown to induce colanic acid production. This is via the activation of the ugd operon which is required for capsule synthesis.
RcsB also upregulates ftsZ which increases the rate of cell division.
enhanced version of ECFP, yeast-optimized
Contains N- and C-terminal linker sequences (with homology to biobrick parts BBa_E2060 (mCherry), BBa_E2050 (mOrange), and BBa_E2030 (Venus YFP) to facilitate color swapping in yeast. Adds N-"MATSG" and "GSGTA"-C. to published amino acid sequence. Inserts a "V" after normal start M. Double TAATAA stop codon. Missing EcoRI, HindIII, NotI, NdeI, XhoI, RsrII, BamHI, NcoI, BglI, SpeI, XbaI, and PstI. Except for 5' and 3' ends no significant sequence identity runs with Venus YFP (BBa_E2030) or to GFP S65T as found in O'Shea deletion strain collection (originally from plasmid pFA6-GFP(S65T)-His3MX6).
Rizzo MA, et al. (2004) Nat Biot (22):445-9
This construct is used to screen the level of induction of the P2 ogr activator on the PF promoter.
PoPS in -> RFP out Strong RBS (efficiency=0.6). Artificial 39 bp terminator (efficiency=0.99).
This is the PO promoter taken from the P2 phage genome. It is an inducible promoter that is activated by a class of activators, including P2 ogr (I746350), PSP3 pag (I746351) and phiR73 delta (I746352). These different activators should cause different levels of activity of the PO promoter.
The source of the DNA is the P2 phage genome.
cinI codes for an autoinducer synthetase which utilizes SAM to make O3-C14:1-HSL. In complex with O3-C14:1-HSL, CinR (BBa_C0077) binds to the Cin promoter (BBa_R0077) and activates transcription.
The regulatory locus cinRI in Rhizobium leguminosarum conrols a network of quorum-sensing loci Lithgow, JK; Wilkinson, A; Hardman, A; Rodelas, B; Wisniewski-Dye, F; Williams, P; Downie, AJ MOL. MICROBIOLOGY 37(1): 81-97, 2000 Change log: original STOP: tga -> tAaTAA
Jay Blumling, Debra Lin, Madeleine Sheldon-dante, Fred Tan, MIT SMUG
This BioBrick is the Gal4 recognition site. It can be used for the construction of promoters which should be regulated by activators or repressors containing a Gal4 DNA binding domain ( BBa_K105007).
This BioBrick contains the consensus sequence plus 5 spacer nucleotides on each side. The spacer is directly followed by the restriction site without a base in between. So when you construct multiple operator sites from this basic part you will have 16 bp between two consensus sequences.
**highly** engineered mutant of red fluorescent protein from Discosoma striata (coral)
monomeric RFP: Red Fluorescent Protein. Excitation peak: 584 nm Emission peak: 607 nm
TAATAA double stop codon added (DE). Four silent mutations made to remove three EcoRI sites and one PstI site: A28G, A76G, A349G, G337A.
Campbell et al., PNAS v99 p7877 URL
downstream construct of the synthetic multicellular bacterium
rbs+glgC (ADP-glucose pyrophosphorylase)
This is the coding sequence of ''glgC'' from ''Escherichia coli'' JM109 with added ribosome binding site (rbs). It encodes ADP-glucose pyrophosphorylase, which catalyses Glucose 1-phosphate + ATP -> ADP-glucose + PPi, a step in the production of glycogen from glucose.
This is the T7 Promoter which is activated when the T7 RNA Polymerase is produced. It provides very good PoPS (Polymerases Per Second) output, as well as it is highly orthogonal to E. Coli's natural transcription system. This one of the moderately active (Mutant D) variations we are using.
Contains both an early MfeI site and a late NsiI site. Cutting with Mfe and Spe and ligation to a BB part cut EX will form two mixed sites, neither of which can be recut. Cutting with Xba and Nsi and ligation to a BB part cut SP will again form two mixed sites, neither of which can be recut.
Should be grown in pSB1A2, as Nsi cuts both pSB2K3 and pSB1AK3 and Mfe cuts pSB4A3. 70 bp from Xba to Nsi or Mfe to Spe, the minimum for gel extraction. The middle is largely noise, to raise the size of the part.
phycocyanobilin:ferredoxin oxidoreductase (PcyA) from synechocystis
Second of two required phycocyanobilin biosynthetic genes.
Coding region for the HK022 bacteriophage cI protein. cI binds to the HK022 pR regulator (BBa_R0050). It represses transcription of the protein encoded by the sequence 3' to the pR region. This coding sequence does not contain a RBS.
References (unparsed) here: Carlson NG, Little JW. Highly cooperative DNA binding by the coliphage HK022 repressor. J Mol Biol. 1993 Apr 20;230(4):1108-30. PMID: 8487297. Mao C, Little JW. Mutations affecting cooperative DNA binding of phage HK022 CI repressor. J Mol Biol. 1998 May 29;279(1):31-48. PMID: 9636698. References (unparsed) here: Carlson NG, Little JW. Highly cooperative DNA binding by the coliphage HK022 repressor. J Mol Biol. 1993 Apr 20;230(4):1108-30. PMID: 8487297. Mao C, Little JW. Mutations affecting cooperative DNA binding of phage HK022 CI repressor. J Mol Biol. 1998 May 29;279(1):31-48. PMID: 9636698. Derived from STHK022N . Response from John Little (Arizona) regarding the start of HK022 cI From: <jlittle@email.arizona.edu> Date: Tue Jan 21, 2003 4:39:21 PM US/Eastern To: "Drew Endy" <endy@MIT.EDU> Subject: RE: hk022 cI start (naive question)? Hello Drew and Michael, I seriously doubt that the extra 27 aa are part of CI. It doesn't make sense in terms of the biology, for sure. In any case, the protein we characterized starts where Carlson and Little stated; as I recall, oR3 partially overlaps the start of cI. I have a vague memory of some possibly interesting biology, having to do with multicopy plasmids. I don't recall the findings themselves. Anyway, one possible explanation was that this extra N-terminal addition was made (e.g. from the pRE promoter, or from a message that arose from transcription around the entire plasmid), making a protein with altered functions. We never followed it up. Good luck John -- Original Message -- Date: Sun, 19 Jan 2003 15:26:26 -0500 Subject: hk022 cI start (naive question)? Cc: elowitm@rockefeller.edu To: jlittle@u.arizona.edu From: Drew Endy <endy@MIT.EDU> Hi John, I'm sitting here with Michael Elowitz and we're working through the sequence for HK022 cI. We noticed that the annotation from NC_002166 includes an "extra" 81 base pairs upstream of what we thought of as the actual start. It looks like this extra DNA extends into the PR regulatory region. We're wondering what's going on here? I'm guessing this is well documented somewhere. Thanks for any pointers/info! Drew It is unknown whether there is cross-talk between this repressor and Lambda cI regulatory region, 434 cI regulatory region and P22 regulatory region.
lasR activator from P. aeruginosa PAO1(+LVA)
coding region for lasR protein, which accepts chemical signal AI-1 (made by lasI protein)
Mutated 480 from g to a to remove PstI site
Genbank: NC_002516 (www.ncbi.nlm.nih.gov)
Lox71 is a site specific recombination cassette. It belongs to the loxP family frequently used in genetics, particularly in mouse genetics. lox site recombination is catalysed by a Site specific recombinase, Cre. lox sequences are composed of an 8 bp Core sequence surrounded by two Arms. The particularity of lox66 is that it has an altered sequence at the end of it's left arm compared to loxP. This sequence variation reduces affinity of the Cre recombinase for the arm. As a consequence, after a recombination between a lox71 and a lox66 (altered right arm sequence), one of the two resulting generated lox sites has very low recombination potential as it inherited both mutated arms. Use of lox71 & lox66 sites is potentially interesting when the recombination reaction must be "irreversible".
No modifaication was made on lox71 sequence
This part was generated in the form of a forward & a reverse primer. After annealing these primers EcoRI & PstI compatible cohesive ends at the 5' & 3' ends of the dsDNA were generated. Next, the dsDNA was subcloned in a pSB1A2 open plasmid (digested with EcoRI & PstI) You can follow the construction process by following the links available in the Paris iGEM 2007 wiki: http://parts.mit.edu/igem07/index.php/Paris "freezer" section plasmids table. A links sends you to the corresponding notebook date when the ligation reaction was performed
Part is composed of TIR domain of Traf6 and GFP linked with 6 aminoacids long linker. GFP serves as reporter. For usage in eukaryotes.
This is the promoter for the ''Eschaerichia coli'' JM109 ''cstA'' gene. It includes the CRP-binding site and the RNA polymerase-binding site. Low glucose concentration results in increased activity by adenylate cyclase. cAMP binds to the cAMP receptor protein, which, in its bound form, is able to associate with the promoter and promote transcription of the downstream gene. (''cstA'' encodes the carbon starvation protein.)
K091146 is a modified version of the pLas' promoter (BBa_K091117) containing the addition of a luxR binding site between the -35 and -10 regions. This promoter is expected to be activated in the presence of PAI-1 and LasR and repressed in the presence of AI-1 and LuxR.
The Lux box was placed adjacent to the LasR binding site with the aim of minimizing cross-talk in the presence of both PAI-1 and AI-1; since the LasR site should have a higher affinity, we hope that the binding of PAI-1 will competitively exclude the binding of AI-1.
The LuxR binding site is from BBa_R0062 and the pLas' promoter is from the region upstream of the LasI gene in Pseudomonas aeruginosa.
pcyA converts biliverdin IXalpha (BV) to phycocyanobilin (PCB). Strong RBS (efficiency=0.6).
Constitutive Promoter, High Transcription
Artificial terminator, estimated %T~=80% # 8bp stem, 6nt loop # Bidirectional, estimated reverse %T~>90%
Bidirectional, with the reverse estimated to be more effective than the forward. Has a polyA tail of 6 residues.
AIP generator + outer membrane permeability
Intermediate - consists of AIP generator (I746000) + term + outer membrane porosity (using the mutant FepA device).
Elowitz RBS. RhlR CDS (no LVA tag) and terminator. Same as I0466 but no LVA tag
Promoter Activity Reporter (LacZ-alpha and GFP-AAV)
mRFP derivative. Ex548nm/Em562. yeast codon optimized.
Contains N- and C-terminal linker sequences (with homology to biobrick parts BBa_E2060 (mCherry), BBa_E2020 (Cerulean CFP), and BBa_E2030 (Venus YFP) to facilitate color swapping in yeast. Adds N-"MATSG" and "GSGTA"-C. to published amino acid sequence. Double TAATAA stop codon. Missing EcoRI, HindIII, NotI, NdeI, XhoI, RsrII, BamHI, NcoI, BglI, SpeI, XbaI, and PstI. except for 5' and 3' ends no significant sequence identity runs to mCherry (BBa_E2060).
[J01008][B0015] ([Key1][Double Terminator])
This is J01008(key 1) with B0015 double terminator in the end.
P2 promoter in agr operon from S. aureus
The agr P2 operon is an autocatalytic sensory transduction system in Staphylococcus aureus. P2 promoter regulates the synthesis of AgrB (a transmembrane protein) and AgrD (precursor of AIP autoinducing peptide). AIP binds to AgrC, a membrane binding receptor and it phosphorylates AgrA. The phosphorylated AgrA increases the expression level from P2 promoter.
Codon usage has been optimised for E. coli (which also seems to give good results for B. subtilis
The section of the sequence constituting the P2 promoter is subject to rather more debate than the sequences of the coding regions. [http://jb.asm.org/cgi/content/full/186/22/7549 Koenig, Robbin L., Ray, Jessica L., Maleki, Soheila J., Smeltzer, Mark S., Hurlburt, Barry K. Staphylococcus aureus AgrA Binding to the RNAIII-agr Regulatory Region J. Bacteriol. 2004 186: 7549-7555] shows the areas of the sequence to which phosphorylated AgrA binds while L Rao, R K Karls, and M J Betley: In vitro transcription of pathogenesis-related genes by purified RNA polymerase from Staphylococcus aureus. Bacteriol. 1995 May; 177(10): 2609???2614. shows the transcription start sites; we took the overall extent of the promoter region from that labelled in Morfeldt, E., K. Tegmark, and S. Arvidson. 1996. Transcriptional control of the agr-dependent virulence gene regulator, RNAIII, in Staphylococcus aureus. Mol. Microbiol. 21:1227???1237. (not available online).
E. coli ars promoter, arsR gene and lacZ
This biobrick contains the promoter region of the Escherichia coli chromosomal arsenic detoxification opern, including the arsR gene encoding the ArsR repressor protein required for inducibility of this promoter. This is fused to a lacZ' minigene encoding the N-terminal 77 amino acid residues of LacZ, sufficient to complement the lacZ-delta-M15 mutation. When introduced into an E. coli host strain carrying the lacZ-delta-M15 mutation, this construct results in lacZ expression in the presence of arsenate or arsenite anion. LacZ expression can be detected in various ways. This construct was designed for the detection of arsenate and arsenite in water samples.
Since this construct includes the arsR gene encoding the ArsR repressor which regulates the ars promoter, it requires no other promoter-specific functions. However, it will only give LacZ activity in a host expressing the E. coli lacZ-delta-M15 gene.
The ars promoter and arsR gene are derived from E. coli JM109 chromosomal DNA. The lacZ' minigene is derived from E. coli BL21 chromosomal DNA, since JM109 does not possess an intact lacZ gene.
Protein generator converting TIPS to the protein TetR. Used as the input section for Quad Part Inverter Q01140.
This is I714031 with R0010 Promoter in the front.
This is the Psid promoter taken from the P4 phage genome. It is an inducible promoter that is activated by a class of activators, including P2 ogr (I746350), PSP3 pag (I746351) and phiR73 delta (I746352). These different activators should cause different levels of activity of the Psid promoter.
''xylE'' is from the ''Pseudomonas putida'' TOL (naphthalene and xylene degradadative plasmid) pWW0. This gene encodes the enzyme catechol-2,3-dioxygenase (metapyrocatechase), which converts catechol to the bright yellow product 2-hydroxy-cis,cis-muconic semialdehyde. This is a useful reporter gene; colonies or broths expressing active XylE, in the presence of oxygen, will rapidly convert catechol, a cheap colourless substrate, to a bright yellow compound with an absorbance maximum around 377 nm. The part includes the native ribosome binding site, so simply has been added to the glucose-repressible promoter of ''cstA''. This allows for characterisation of pCstA over a range of glucose concentrations.
The template DNA for ''xylE'' was kindly supplied by Dr. Peter Williams of the University of Wales, Bangor. pCstA is from ''Escherichia coli'' JM109 genomic DNA.
LuxI regulated by the pLac promoter. Used for expressing an adjustable amount of LuxI.
mnt repressor (weak) from Salmonella phage P22 (+LVA)
dimeric repressor from enterobacteriophage p22 with dissociation constant of 3*10^-8. sequence was translated with the E.Coli K12 codon usage table, and nucleotides 95-97 was changed from AAT to AAC to avoid the cut site GAATTC (94-99). the mutation R10->K10 (ctg->aaa) decreases the binding affinity of the mnt protein to its operator by approximately 12,000 fold (Kd: 2.5e-12 to 3e-8)
Maltose binding protein fused to Zif268-GCN4.
Double terminator consisting of BBa_B0010 and BBa_B0012
mnt repressor (strong) from Salmonella phage P22 (+LVA)
wild type dimeric repressor from enterobacteriophage p22 with dissociation constant of 2.5*10^-12. sequence was translated with the E.Coli K12 codon usage table, and nucleotides 95-97 was changed from AAT to AAC to avoid the cut site GAATTC (94-99). Identification of Functionally Important Residues in the DNA Binding Region of the MNT Repressor Kendall, KL; Sauer, RT The Journal of Biological Chemistry, 264(23):13706-13710, 1989
Constitutive Promoter, Medium Transcription
Srini Devadas, David Gray, Ronny Krashinsky, Debra Lin, and Chris Zheng Liu
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.
pBAD to tetR coding region, lacI coding region, and mCherry reporter
Links the pBAD promoter, which is induced by arabinose and inhibited by araC, to a tetR coding region with an RBS, a LacI coding region with an RBS, and an mCherry reporter. Activation of this pathway results in tetR production, lacI production, and mCherry production.
Strength of RBS may affect the strength of output.
This part can generate the lacI repressor. Normally, however, the production of lacI is inhibited by the presence of the lock. But when key is added, lacI can be generated.
PenP operator from Bacillus Lichenformis. Two operator sites are negatively regulated by PenI (C0074).
enhanced cyan fluorescent protein derived from A. victoria GFP
Artificial terminator, estimated %T~<10% 8bp stem, 6nt loop Bidirectional, estimated reverse %T~=40%
Bidirectional, with the reverse estimated to be more effective than the forward. Has a polyA tail of 3 residues.
autoinducer synthetase for PAI from Pseudomonas aeruginosa
coding region for lasI protein, which produces the chemical signal AI-1
Binding region for LasR protein (positive regulation)
"Analysis of the Pseudomonas aeruginosa Elastase (lasB) Regulatory Region". Lynn Rust, Everett Pesci, and Barbara Iglewski
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.
OriT-R (Origin of Transfer for the R751-plasmid nic region)
OriT-R751 (Origin of Transfer for the R751-type conjugative plasmid) is the nic region, where the relaxosome nicks the plasmid and conjugative transfer by R751 plasmid machinery begins via rolling circle replication.
Enterobacter aerogenes plasmid R751 NCBI:NC_001735
Coding SSM device: GFP with (AGTC)10 after start codon
Strong promoter + strong RBS + GFP with (AGTC)9 repeat after start codon + double terminator
This part has been derived from the BBa_J22101 part. The BBa_J22101 part was not working due to an insertion of 35 bp upstream the LacY coding sequence. We amplified the right gene sequence by PCR and put it into a standard plasmid.
This is the T7 Promoter which is activated when the T7 RNA Polymerase is produced. It provides very good PoPS (Polymerases Per Second) output, as well as it is highly orthogonal to E. Coli's natural transcription system. This one of the moderately active (Mutant E) variations we are using.
The lysozyme from enterobacteria phage T4 degrades peptidoglycan layer.
High expression of lysozyme only is toxic to bacteria.
Enterobacteria phage T4 http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=29366675&from=66503&to=66997&view=gbwithparts
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.
Regulatory SSM device: Promoter with (TA)9 repeat expressing GFP
Promoter with (TA)9 repeat upstream of RBS + GFP + TT
This device can generate GFP, but the production of GFP is normally switched off. Only when key is present, GFP will be produced. In that case fluorescence is detectable.
TP901 cI repressor from phage TP901-1 (+LVA)
Dimeric repressor from bacteriophage TP901-1 with dissociation constant of 2*10^-9. Sequence was translated with the E.Coli K12 codon usage table, and nucleotides 84-86 was changed from TTC to TTT to avoid the cut site GAATTC (81-86). Bronsted, L., Hammer, K., Johansen, A.H., 2003. Identification of operator sites of the CI repressor of the phage TP901-1: evolutionary link to the other phages. Virology 311, 144-156. cI sequence slightly different from part C0051. NCBI locus: NC_001416
Modificaitons to Clontech EYFP by Reshma Shetty
Removed LVA tag from BBa_C0052 via PCR. In pSB1AK3 currently.
Lac repressible lasI generator (No LVA )
Masahiro Tominaga,Yoshimi Iyama,Kohei Kawasaki
Generates 3OC12-HSL under a lac-repressible promoter
cI repressor (+LVA) regulated by pLac Promoter
Coding region for the cI repressor (C0051) 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). The expression of the protein is under the regulation of pLac Promoter (R0010).
Reporter construct for constitutive YFP and inducible CFP
Reporter Construct with Tet operator+YFP(LVA-) reporter followed by a Lac operator +CFP(LVA-) reporter
In strains with lacZ-omega (lacZ N-terminal deletion mutant) like DH5alpha, DH10B and Top10, lacZ-alpha restores the beta-galactosidase activity.
PCR amplified from BBa_J33202 without the RBS.
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).
GFP expression controlled by LuxR promoter
This forms a GFP coding region which is controlled by the pLuxR promoter. This part will be used to form J37020, a test construct for the polycistronic predator cell (Imperial College iGEM 2006)
Parts ligated from existing parts in registry
lasR activator from P. aeruginosa PAO1(no LVA)
PoPS in -> luxI out. Strong RBS (efficiency=0.6). Artificial 39 bp terminator (efficiency=0.99).
COMT coding sequence Encodes caffeic acid-O-methyl transferase (methylates -OH on C4 of the aromatic ring) from Medicago sativa (alfalfa), synthesised by Geneart
Used cDNA as coding gene in Alfalfa genome contains introns.
Alfalfa cDNA Genes and Enzymes Involved in Caffeic Acid Biosynthesis in the Actinomycete Saccharothrix esanaensis, Berner M, et al, Journal of Bacteriology 188(7): 2666, (2006) Gowri G, et al, Molecular cloning and expression of alfalfa S-adenosyl-L-methionine: caffeic acid 3-0-methyltransferase, a key enzyme of lignin biosynthesis, Plant Physiology 97(1):7 (1991) Downregulation of Caffeic Acid 3-O-Methyltransferase and Caffeoyl CoA 3-O-Methyltransferase in Transgenic Alfalfa Impacts on Lignin Structure and Implications for the Biosynthesis of G and S Lignin, Guo D, et al, Plant Cell. January; 13(1): 73 (2001)
PCR sil03F and sil03R on barnase region (348 bp, BglII/XhoI) Sub into pBca9145-Bca1089 (BglII/XhoI, 2063+696, L) Product is pBca9145-I716211 sil03F Forward BglII biobrick of barnase ccaaaAGATCTgcacaggttatcaacacgtttg sil03R Reverse XholI/BamHI biobrick of barnase gctagCTCGAGttaGGATCCttatctgatttttgtaaagg
SREBP regulated promoter, predicted, tested HB8
This is a two-protein generator (cI and lasR) without transcriptional terminator. Strong RBS (efficiency=0.6).
glgC coding sequence encoding ADP-glucose pyrophosphorylase
This is the coding sequence of ''glgC'' from ''Escherichia coli'' JM109. It encodes ADP-glucose pyrophosphorylase, which catalyses Glucose 1-phosphate + ATP -> ADP-glucose + PPi, a step in the production of glycogen from glucose.
The genomic DNA sequence contains to EcoRI sites, both of which have been mutated out using the mutagenesis by blunt-ended ligation (MABEL) technique
CheB chemotaxis coding sequence (protein glutamate methylesterase)
CheB is part of the chemotaxis pathway in E. coli.
Checked (DE, VC, KN). Part ready for synthesis. 8/3/4
NCBI - E. Coli strain K12 substrain MG1655
luxI protein synthesizes 3OC6HSL, which binds to LuxR. Strong RBS (efficiency=0.6).
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
Modified lamdba Prm promoter (repressed by 434 cI)
Lamdba Prm promoter modified to be activated by lamda repressor (cI) and repressed by 434 repressor (cI)
The O-R1 region of 434 contained 14 base pairs as opposed to the 17 base pairs of the O-R3 site of lambda. Also, it was noticed that the O-R3 site of the lambda included part of the -10 site. Hence, to preserve the spacing and the -10 site, the three nucleotides that were in both the -10 site and the lambda O-R3 site were retained. The 14 nucleotides that were in the O-R3 site and not in the -10 site were replaced with the O-R1 site of the 434.
3OC6HSL Sender Controlled by Lac Repressible Promoter
A lac repressible promoter driving production of an untagged LuxI enzyme which produces an AHL (3OC6HSL).
This promoter is repressed by lacI and luxR with HSL. It contains a lux box and a lacI binding site.
R0010 modified with the insertion of a lux box in between -35 and -10.
crRNA-lock3-var1 (A new "lock" designed by our team)
crRNA(I714033) and taRNA(I714034) whose structure resemble that of J23078 (lock3) and J23066 (key3) but with a different matching regions to minimize the possible interaction with the key3-lock3.
LuxR regulated by the pTet promoter. Used to constitutively express LuxR.
FILTER This device was designed to filter out noise in an input signal; in our case OmpF PoPs. If there is a short increase in transcription from this promoter, a small amount of J23066 RiboKey is produced. This is then used to unlock (J23078) the short lived UmuD-tagged T7 RNA polymerase. This results in an increase of active T7 protein. The final part of this filter is an AND gate which is used in the components further on in the system. This AND gate consists of a T7 RNA polymerase promoter with another RiboLock (J23078 again). The AND gate will only function if the duration of the noise is sufficiently long and intense (= a signal) in order to have both the short-lived RiboKey AND T7 polymerase available at the same time and thus to produce the component.
We did a lot of modeling on this device. The most efficient Berkeley 2006 key and lock have been used. The RiboKy might be required in higher copy number although modeling doesn't predict this.
cinR activator from Rhizobium leguminosarum (+LVA)
In complex with O3-C14:1-HSL (made by BBa_C0076), CinR binds to the Cin promoter (BBa_R0077) and activates transcription.
The regulatory locus cinRI in Rhizobium leguminosarum conrols a network of quorum-sensing loci Lithgow, JK; Wilkinson, A; Hardman, A; Rodelas, B; Wisniewski-Dye, F; Williams, P; Downie, AJ MOL. MICROBIOLOGY 37(1): 81-97, 2000 Change log: original STOP: tag -> taATAA
Regulatory SSM device: Promoter with (TA)10 repeat expressing GFP
Promoter with (TA)10 repeat upstream of RBS + GFP + TT
BBa_J04800 (RevAptRibo) Riboswitch with BBa_J04671 (EYFP, RBS-, LVA+, B0015)
This is a (RevAptRibo) Riboswitch device that contains a EYFP reporter region with an LVA tag and a B0015 Terminator Sequence
downstream construct of the synthetic multicellular bacteria
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.
LsrA promoter (indirectly activated by AI-2)
The regulatory network for the uptake of Escherichia coli autoinducer 2 (AI-2) is comprised of a transporter complex, LsrABCD; its repressor, LsrR; and a cognate signal kinase, LsrK. This network is an integral part of the AI-2 quorum-sensing (QS) system. Once uptaken into the cell, AI-2 will be phosphorylated under the effect of LsrK. Without the presence of AI-2, LsrA promoter is inhibited by the repressor, LsrR. Phosphorylated AI-2 will prevent LsrR from inhibiting this promoter, hence activating it so that the downstream sequence can be expressed. To prevent the cell from self-inducing this promoter, we must suppress its AI-2 producing capability. It can be done by knocking out LuxS, one of the vital gene required for producing AI-2. By doing that, LsrA promoter can only be activated under the presence of AI-2 from other sources than the host cell which is LuxS mutant (say, by contact with normal cells). More information on AI-2 quorum-sensing (QS) system please refer to: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1952038
Rluc-linker-PEST191 is a part composed of Rluc (which acts as a reporter) linked with PEST191 through six aminoacid long protein linker. PEST191 is a sequence that leads towards degradation of protein linked with this sequence.
Rluc-linker-PEST 191 was made by using PCR ligation technique. This part is cloned in BioBrick vector pSB1AK3.
Site directed mutagenesis was carried out to change a glutamine residue to a serine to make the PAH enzyme protease resistant. This will allow safe passage through the stomach and gut in order to carry out function as required by Imperial College's 2009 iGEM project, The E.ncapsulator.
Site directed mutagenesis was carried out to obtain the desired characteristics
PLac activation induces the expression of the Lactose permase (Lac Y) gene, LacI repressor protein with GFP (+LVA tag) as a reporter.
This construct is used to screen the level of induction of the phiR73 delta activator on the PLL promoter. Note that the I746352 does contain a RBS.
PelB leader sequence; directs protein to E. coli periplasmic membrane
The pelB leader sequence is a sequence of amino acids which when attached to a protein, directs the protein to the periplasmic membrane of E. coli, where the sequence is removed by pelB peptidase. It is used to direct coat protein-antigen fusions to the cell surface.
Links an lacI coding region with an RBS to mCherry reporter
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.
Lysis gene (promotes lysis in colicin-producing bacteria strain)
This lysis gene encodes for the lysis protein in colicin-producing strains of bacteria. Once activated, it causes the host cell to lyze. It also removes the immunity protein out of colicin, and hence, activates the endonuclease activity of the colicin.
Digest pBca9145-I716206 (BamHI/AlwnI, 1694+553bp, Large) Digest pBca9145-Bca1114 (BglII/AlwnI, 1519+593bp, Small) Product is pBca9145-I716208.
intermediate for lac relaxation oscillator
intermediate part for the lac version of the synchronized relaxation oscillator
Sequence verified, however there is an N at position 1021, however upon looking at the actual sequence trace it looks like it is most likely the correct base.
crtZ (beta-carotene hydroxylase) with native rbs
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.
Pantoea ananatis (formerly Erwinia uredovora) DSMZ 30080 (ATCC 19321). Accession: D90087.
Promoter, Standard (lambda cI regulated)
Vinay S Mahajan, Brian Chow, Peter Carr, Voichita Marinescu and Alexander D. Wissner-Gross Drew Endy
[Note: This is the same part as R0051 except that the -10 and -35 sites and spacing have been changed to comply with BBa_S0001]. The cI regulated promoter is based on the pR promtoer from bacteriohage lambda. The promoter has two two DNA binding sites for lambda cI repressor <bb_part>BBa_C0051</bb_part>. cI binding results in repression of transcription. The specific sequence used here is based on the cI repressible promoter used in the Elowitz repressilator (and references therein).
In order to address concerns about the promoter transcribing in the reverse direction, we have removed the -35 and -10 signals responsible for the promoter activity in the reverse direction. (More details needed here! DE, 2/24/03) Modified to comply with BBa_S0001: TTGACA-17N-GATACT Incompatible with host expressing cI repressor.
A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000
GFP reporter protein under the control of the J23100 constitutive promoter and LexA 2 operator
The second screening plasmid. Same as the first, except with stronger RBSs. Ends should be capped with I13450.
Inhibitor (AraC loop attachment with O2 site)
this piece of DNA contains a "dead" part of coding region for AraC, and a functional Operator2 site. when attached to the rest of the promoter region, this part allows for the binding and loop formation that would result in repression of the araC promoter
1) changed ATG start codon to TAG stop 2) 6 random deletions upstream of putative araC binding sites to preserve spacing for loop back (BB ends will add 6 base pairs); spacing crucial in matching up O2 and I2 binding sites (turns of DNA helix) 3) designed to attach to R0080 to enable all (positive and negative) regulatory functions, and to form "loop back"
GFP fimE switch with 250 bp tetR promoter
A promoter facing upstream is flanked by two FimE binding sites. GFP is downstream to this fimE switch. Active FimE will flip the promoter such that GFP is expresssed.
PCR sil04F and sil04R on BamHI methylase/endonuclease region. (657 bp, BglII/XhoI) Sub into pBca9145-Bca1089 (BglII/XhoI,2063+696, L) Product is pBca9145-I716210 sil04F Forward BglII biobrick of BamHI ccaaaAGATCTgaagtagaaaaagagtttattactga sil04R Reverse XholI/BamHI biobrick of BamHI gctagCTCGAGttaGGATCCctatttgttttcaactttatc
PCR sil04F and sil04R on BamHI methylase/endonuclease region. (657 bp, BglII/XhoI) Sub into pBca9145-Bca1089 (BglII/XhoI,2063+696, L) Product is pBca9145-I716210
rbs+glgC16 (glgC with G336D substitution)
This is the coding sequence of ''glgC'' (ADP-glucose pyrophosphorylase) from ''Escherichia coli'' JM109 with the substitution G336D, with added ribosome binding site (rbs). This mutation is known to cause increased activity of ADP-glucose pyrophosphorylase in the absence of the activator fructose 1,6-bisphosphate (FBP), high affinity for FBP and substrates lower affinity for the inhibitor AMP. (Leung ''et al.'', 1986; Meyer ''et al.'', 1988)
This construct is used to screen the level of induction of the phiR73 delta activator on the Psid promoter. Note that the I746352 does contain a RBS.
This BioBrick is the lex A recognition site. It can be used for the construction of promoters which should be regulated by activators or repressors containing a lex A DNA binding domain ( BBa_K105005).
The fourth screening plasmid. Same as the second, except without an insertion site. Sole purpose is to check whether the insertion site alone affects the performance. Ends should still be capped with I13450.
cI protein generator without terminator (strong RBS, efficiency=0.6)
cI: Vinay S Mahajan, Brian Chow, Peter Carr, Voichita Marinescu and Alexander D. Wissner-Gross RBS: Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
Artificial terminator, estimated %T~>90% * 8bp stem, 6nt loop * Bidirectional, estimated reverse %T~>90%
Bidirectional, with the reverse estimated to be more effective than the forward. Has a polyA tail of 4 residues
XylR Transcriptional Regulator Right Facing
Code for XylR Transcriptional regulator of xylose catbolization pathway. XylR recognizes xylulose (the isomer of xylose) and promotes the transcription for the high affinity transporter (XylF,G,H) and catbolization enzymes XylA,B.
This is the PF promoter taken from the P2 phage genome. It is an inducible promoter that is activated by a class of activators, including P2 ogr (I746350), PSP3 pag (I746351) and phiR73 delta (I746352). These different activators should cause different levels of activity of the PF promoter.
no special considerations seemed necessary
The original source is the P2 phage genome. For our work the DNA came from plasmids, kindly supplied to us by Prof. Calendar, University of California (Berkeley): Bacteriophage PSP3 and fR73 Activator Proteins: Analysis of Promoter Specificities; Julien and Calendar, 1996; JOURNAL OF BACTERIOLOGY, Oct. 1996, p. 5668???5675
This is the coding sequence of crtB from Pantoea ananatis (formerly Erwinia uredovora) (Accession number D90087) preceded by an E. coli synthetic ribosome binding site (rbs). It encodes phytoene synthase, part of the carotenoid biosynthesis pathway, which converts geranylgeranyl diphosphate to phytoene.
rbs: Escherichia coli JM109 genomic DNA. crtB: Pantoea ananatis genomic DNA
This is the coding sequence of ''glgC'' (ADP-glucose pyrophosphorylase) from ''Escherichia coli'' JM109 with the substitution G336D. This mutation is known to cause increased activity of ADP-glucose pyrophosphorylase in the absence of the activator fructose 1,6-bisphosphate (FBP), high affinity for FBP and substrates lower affinity for the inhibitor AMP. (Leung ''et al'', 1986; Meyer ''et al.'', 1988)
The mutation G336D was achieved by the substitution of A for G at position 1076 (codon GGC->GAC) by MABEL.
The SupD-tRNA part functions as a tRNA, it suppresses the TAG amber stop codon and translate it into a Ser. There is no need to assembly SupD-tRNA with a rbs, since it is not translated. This part, together with the T7ptag part, make an AND Gate, that is, in the presence of both the 2 parts, T7 polymerase is tranlated into a functional polymerase that transcrips a downstream element constructed after a T7 promoter. Which is the output of the AND Gate.
There is a PstI site in the original sequence, but luckily it is near the 3' end of this part, so we designed a mutation in our primer and mutated the PstI site.
SupD-tRNA again is from Voigt Lab(UCSF), We standardlize this part by PCR strategy.
enhanced version of EYFP, yeast-optimized
Venus YFP, yeast codon optimized. EX515/EM528
Contains N- and C-terminal linker sequences (with homology to biobrick parts BBa_E2060 (mCherry), BBa_E2050 (mOrange), and BBa_E2020 (Cerulean CFP) to facilitate color swapping in yeast. Adds N-"MATSG" and "GSGTA"-C. to published amino acid sequence. Inserts a "V" after normal start M. Double TAATAA stop codon. Missing EcoRI, HindIII, NotI, NdeI, XhoI, RsrII, BamHI, NcoI, BglI, SpeI, XbaI, and PstI. Except for 5' and 3' ends no significant sequence identity runs with Cerulean CFP (BBa_E2020) or to GFP S65T as found in O'Shea deletion strain collection (originally from plasmid pFA6-GFP(S65T)-His3MX6).
Reporter Construct with Tet operator+YFP(LVA+) reporter followed by a Lac operator +CFP(LVA+) reporter
Weaker version of wild type pBAD ( I13453 ).
Site-directed mutagenesis on I13453 with the following primers: Forward: TAATCTTATGGATAAAAAAGCTATGGCATAGC Reverse: GCGGATCCTACCTGACGCTTTTTATC
OriT-R (Origin of transfer for the R-plasmid nic region)
OriTR, the R plasmid nic region, is where the relaxosome nicks the plasmid and conjugative transfer by R plasmid machinery begins.
Pr promoter of the lambda-system combined with GFP as a quantification and debugging device for the INPUT-module of the ETHZ05 iGEM team.
For more details see Sensing Device and Event Processing Device documentation.
The kozak sequence acts as a eukaryotic RBS. It is cloned directly between a promoter and coding region.
This part accepts POPS to produce ECFP under the medium RBS B0032
penI repressor from Bacillus licheniformis (+LVA)
Since penI is being ported from Bacillus Licheniformis, the sequence was changed within the coding region to reflect codon usage in E.Coli K12. Codons were mapped to the codon of the same usage rank in E.Coli.
This part includes the xylE gene from the Pseudomonas putida TOL (naphthalene and xylene degradadative plasmid) pWW0. This gene encodes the enzyme catechol-2,3-dioxygenase (metapyrocatechase), which converts catechol to the bright yellow product 2-hydroxy-cis,cis-muconic semialdehyde. This is a useful reporter gene; colonies or broths expressing active XylE, in the presence of oxygen, will rapidly convert catechol, a cheap colourless substrate, to a bright yellow compound with an absorbance maximum around 377 nm. The part includes the native ribosome binding site, so simply needs to be added after a suitable promoter to act as a reporter. I have previously used this gene to generate whole cell biosensors for various heavy metals. Note that, unlike Xgal etc., catechol in solution is prone to spontaneous oxidation resulting in brown melanin-like polymeric products, so is not stable enough to incorporate into plates or growth media; it should be dripped onto colonies (I use 10 mM catechol in water for this) or added to liquid cultures at a final concentration of about 0.5 mM prior to assay. Note also that there is a SacI site at the very start of the part, when the prefix is included, so this can be used as a replacement vector to introduce PCR products with SacI-SpeI ends into pSB1A2 giving them full Biobrick prefixes and suffixes (but don't forget the G base before the SpeI site). This results in shorter non-complementary tails on PCR primers than using a full prefix or suffix. You can test for colonies that have lost xylE using catechol as described above.
Note that this sequence includes the native ribsomome binding site. Also, when the prefix is included, there is a SacI site at the start, which allows this part to be used as a vector for insertion of PCR products with SacI-SpeI ends into pSB1A2, replacing xylE, giving the full Biobrick prefixes and suffixes (but don't forget the G base before the SpeI site). This results in shorter non-complementary tails on PCR primers than using a full prefix or suffix. You can test for colonies that have lost xylE using catechol as described above.
The template DNA was kindly supplied by Dr. Peter Williams of the University of Wales, Bangor. The primer design was based on Genbank sequence M64747 (GI:151718). The sequence reported here was confirmed by sequencing the Biobrick construct.
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.
QPI Test Construct Intermediate (Q01400.E0430)
Quadpart Amplifier (B0034.C0062.B0015.R0062)
Upstream construct of the Synthetics Multicellular Bacterium
I746000 (AIP generator - agrB + agrD with RBSes) + B0015 (Terminator)
This part is simply the AIP generator from the agr system with a standard registry terminator downstream. The original (I746000) 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 agrB and agrD were taken from the S. aureus Sequencing Project 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.
E. coli chromosomal ars promoter with arsR repressor gene
This part consists of the promoter of the E. coli JM109 chromosomal arsenic detoxification operon (ars operon), including the ArsR repressor binding site and the arsR gene encoding the arsR repressor protein, together with its ribosome binding site. Addition of any other genes to the 3' end of this part will result in their expression being dependent on the presence of sodium arsenate or sodium arsenite. Arsenite or arsenite anion binds to the repressor protein ArsR, resulting in inability to repress the promoter. Based on our experiments, a concentration of 1 micromolar sodium arsenate in LB is sufficient for essentially full expression, though this will vary according to conditions.
Note that this sequence includes the arsR gene encoding the ArsR repressor protein, which is thus negatively autoregulated. No additional parts are required for arsenate-induced expression from this part. In principle, this part should also function in hosts other than E. coli.
The DNA was derived by PCR using genomic DNA from E. coli JM109. Primers were designed based on the sequence reported by Diorio et al (1995), J. Bacteriol 177 (8), 2050-2056, Genbank accession X80057, GI:510824. The sequence given here is derived by sequencing the biobrick construct.
This part is used for purification of P7 GFP (for source and other information about this new GFP variant see its part description: I746917) via its 6-his tag. It is driven by a T7 promoter. We used E.coli BL21 (DE3) for expression of this part: addition of 1mM IPTG leads to expression of the T7 DNA polymerase which in turn drives expression of the tagged P7 GFP.
The part was created from intermediates via standard assembly.
Bidirectional transcriptional terminator cloned in the reverse direction of B0011. 22 bp stem-loop
Contains an IPTG inducible promoter an RBS, RFP, and terminator.
Construction intermediate: Lambda cI QPI with EYFP (Q04510.E0430)
Elowitz RBS. CinR CDS (no LVA tag) and terminator. Same as I0468 but no LVA tag
BBa_J04900 (3AptRibo) Riboswitch with BBa_J04671 (EYFP, RBS-, LVA+, B0015)
This is a (3AptRibo) Riboswitch device that contains a EYFP reporter region with an LVA tag and a B0015 Terminator Sequence
autoinducer inactivation enzyme aiiA (no LVA)
This protein is part of a two part signaling system. Phosphorylation of ComA by ComP(a cell surface receptor) results in its activation. It then initiates transcription from a specific response promoter. This protein is not endogenous to E. coli.
Synthesized based on genomic sequence in B. subtilus
Tet repressor generator (version1, TERM+)
There is a mutation induced by PCR in [http://partsregistry.org/wiki/index.php/Part:BBa_I732084 BBa_I732084]. But we are convinced that the mutation has nothing to do.
excisionase from E. coli phage P22 (removes prophage from host genome)
PoPS in -> pcyA out. Strong RBS (efficiency=0.6). Artificial 39 bp terminator (efficiency=0.99).
Modified lambda Prm promoter (OR-3 obliterated)
Lambda Prm promoter modified to be activated but not repressed by the lambda repressor (cI)
In wild type lambda phage, the OR3 site (-27 to -11) reads "tatcccttgcggtgata" on the sense strand of DNA. To prevent lambda repressor (cI) from binding to this site, the 4th through 10th nt of OR3 were replaced by the 7 nt between OR2 and OR1, "aaatagt" (-57 to -51), which in effect mutates 5 nt of the promoter. These nt were chosen to be about halfway between the -10 and -35 boxes of the promoter. Furthermore, since these nt already act as a spacer in wild-type phage, it is hoped they will not create undesired interactions.
This part is designed to ensure that we can detect both RFP and GFP in the same cell.
Artificial terminator, estimated %T~=25% * 6bp stem, 4nt loop * Bidirectional, estimated reverse %T~<10%
Bidirectional, with the reverse estimated to be less effective than the forward. Has a polyA tail of 3 residues. Designed to be a terminator with low efficiency.
alcohol acetyltransferase I; converts isoamyl alcohol to isoamyl acetate (banana odor)
This part is the ATF sequence (BBa_45J006) with a single point mutation at base pair 1176 (T -> C).
It has a single point mutation at base pair 1176 (T -> C).
Charles Fracchia, Royah Vaezi, Tianyi Wang, Nuri Purswani
Phenylalanine hydroxylase (PAH) is an enzyme used to break down phenylalanine in humans.
araC arabinose operon regulatory protein (repressor/activator) from E. coli (+LVA)
coding region for the araC gene, used to make araC protein for use in positive or negative regulation of TIPs output (see also R0080, R0081)
GenBank: NC_002655 (www.ncbi.nlm.nih.gov)
autoinducer synthetase for N-butyryl-HSL (BHL) and HHL
Autoinducer synthesis protein that produces N-butyryl-HSL which binds to RhlR, obtained from Pseudomonas aeruginosa.
Editing Check (1/28/04) (0) BB sites cleaned (1) ATG (2) TAATAA (3) Blast checks out (i.e., it's RhlI) (4) Codon changes OK (AA and tRNA usage) (5) Chassis might have conflict (w/ native autoinducer system?) (6) ssrA tag added (7) barcode added (8) Codon optimized for expression in enteric bacteria 2 silent point mutations have been included into the sequence at base 138 from A to G and at base 576 from G to C to remove internal EcoRI and PstI sites. Mutations were chosen to yield the same amino acid and codons of similar frequency in E. coli.
This is an enzyme involved in the heme biosynthetic pathway. Also called uroporphyrinogen III synthase.
This part results from ligating E1010 with B0015 in the manner described on this website.
This part is used to screen the level of induction of the PF promoter (I746360). The PF promoter is induced by a class of phage activators (P2 ogr: I746350, PSP3pag: I746351, phiR73 delta: I746352).
The PF promoter was put upstream of I13504.
registry (I13504) and own construct (I746360)
Deletion of the C at position 1194. This is right at the end of I0500, after the transcriptional start of the downstream gene but before the RBS. Shouldn't make a difference, but needs to be checked out.
promoter lambda (cI regulated) with RFP reporter
Promoter lambda - cI regulated - (R0051) with RFP (E1010) as a reporter. It can be used as a detector of the glucose metabolism.
This is a universal part designed to allow induction by HSL of whatever is assembled downstream of the part.
This part was constructed from registry biobricks.
Upstream construct of the synthetic multicellular bacteria
Intermediate part composed by RBS (B0034), GFP with LVA tag and terminators.
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