marR(G95S) carries a G>A mutation at position 284 which gives a amino acid change G95S. Has been mutated from BBa_K864200.
A mutant of the ERG20 gene; K197E substitution. The encoded mutant protein produces 25% FPP and 75% GPP instead of 75% FPP and 25% GPP compared to the wild type Erg20. GPP is a precursor in producing monoterpenes whereas FPP produces diterpenoids instead. Reference: Oswald M., Fischer M., Dirninger N., Karst F. Monoterpenoid biosynthesis in Saccharomyces cerevisiae. 2007.
tRNA(Asp) with its anticodon mutated to CUA (base pairing stop codon UAG) and under the control of lpp promoter. This biobrick is constructed first by cloning the tRNA(Asp) from AspV in E.coli, then the anticodon region is site-directed mutated.
the correct processing of tRNA should be ensured
pBAD+Theophylline Responsive Riboswitch 1G1 with Engineered RBS+E0040+B0015
It is a theophylline responsive riboswitch which contains a designed RBS.
cheZ, chemotaxis regulator, protein phosphatase for CheY
Polar localization is dependent on CheA-short. CheZ is a cytosolic phosphatase which functions in the chemotaxis signal transduction complex. Plays an important role in bacterial chemotaxis signal transduction pathway by accelerating the dephosphorylation of phosphorylated CheY (CheY-P).
how to control the proper expression of cheZ
This composite part constitutively expresses a ToxR-LambdaRep construct. This ToxR construct will dimerize at a high affinity, because of Lambda Rep's dimerizing properties, making the cytoplasmic ToxR dimer an active transcription factor for the ctx Promoter. Thus, If this composite part is combined with a Pctx-GFP reporter there will be a high expression of GFP.
The biggest design considerations were how to limit the amount of ToxR expressed while still allowing enough ToxR to be produced to see a high expression of GFP. The P_rffGH promoter, the rbs, and the plasmid we put it on (ColE1 origin) all contributed to the optimal expression level of the ToxR construct.
The original promoter came from E. Coli genome, but this specific promoter was selected from a library of that promoter. This rbs was selected from a library in a tuning process. ToxR comes from a virulence island of Vibrio Cholerae. The Lambda Repressor is from the Lambda Phage.
Lambda cI and LuxR regulated - hybrid promoter + GFP generator
Bakul Jitendra Vinchhi, Sarah Veugelen, Katrien Vandermeeren, Yana Hoorne
This part was designed to test the functionality of the promotor region.
promoter and RBS of E. coli chitoporin translationally fused with lacZ.
ChiP encodes enterobacterial chitoporin required for uptake of chitin-derived oligosaccharides. This outer membrane porin is required for Salmonella and E.coli growth on chitooligosaccharides as a sole source of both carbon and nitrogen. It is only synthesized in the presence of these substrates. In nature it is regulated by sRNAs.The upstream part of this gene is fused with reporter gene lacZ to allow checking if the regulation works correctly.
The subparts were prepared: ChiP upstream part from Escherichia Coli W3110 genomic DNA by PCR and lacZ from part BBa_I732005 present in part registry.
Modified 3' "half" of intron found in ORF #142 in Twort phage of Staphylococcus aureus in RFC53 MetaPart format. When complemented with 5' "half" ( BBa_K576004 ) in complience with BBFRFC53 assembly standard, the RNA-level excision should leave GGU linker between the whatever comes before K576003 and after K576004. K576003 and K576004 and everything in between them would be removed and possibly circularized.
The P1 helix of the intron has been modified to accommodate the MetaPrefix. The part is in RFC53 L-MetaPart format, having GGT and ATG overgangs.
[http://www.rna.whu.edu.cn/perl/gissd/get_seq_exon.cgi?intron_id=304 Group I intron Twort.v.ORF142,2]
James Coletta, Anisha Katyal, Kristen Salavo, Lauren Rossi
The operating region for the Lambda switch that represses C1 and Cro bind to. This region contains promoters prm and pr facing in opposite directions. It also contains operating regions Or3, Or2 and Or1. In the lysogenic state, the C1 dimer is bound naturally bound to the Or1 repressing promotor pr and production of cro. Once RecA cleaves enough C1 dimer the system switches to the lytic state. The Cro produced at the pr promoter then binds to the Or3 site inhibiting the prm promoter. For a better description of the Lambda system read the book "A genetic switch" by Mark Ptashne. We used Or in creation of the lambda system, as part of our project to create a sensor for DNA damage in bacteria due to radiation.
We constructed this part by combining K649000 and K081008. This part starts generating LuxI enzyme in the presence of LasR-3OC12HSL complex.
Amplified from MG1655 (accession NC_000913) chromosome using primers listed below (annealing sequences are underlined) lexA_BB_F TTCGAATTCGCGGCCGCTTCTAGATGAAAGCGTTAACGGCCAG lexA_BB_R CGGCCGCTACTAGTATTATTACAGCCAGTCGCCGTT
Parts K611021 through K611027 are variants of the wild type LacI repressible promoter, BBa_R0010. These parts were generated through three rounds of mutagenic PCR and each have around 1 to 7 mutations. Each of these variants differ in properties such as transcriptional strength and repressor binding affinity and have been well characterized. This can be very useful in tuning genetic circuits for specific outputs.
We chose to mutate this part from a well known promoter so that any changes in behavior would be easy to observe.
This is a version of BBa_K523002 but with no RBS and no stop codon. There are four extra bases at the end which (together with the standard RFC10 prefix) generate a BglII site. There are two extra bases at the end which, when combined with the T at the start of the standard RFC10 suffix, code for a glycine. The effect of both of these features is to provide an in-frame BglII site at the start, and an in-frame SpeI site at the end (from the standard suffix). These features are necessary for Edinburgh 2011's BioSandwich assembly protocol.
The part was made using the strategy outlined in BBa_K523000 , and therefore contains 4 extra bases at the 5' end which generate a BglII restriction site. The part was created from a lab ''E. coli'' strain using primers: * ACT AGATCT atg aaa tgg cta tgt tca gta gg ** Forward; adds BglII site; starts at start codon. * ACT ACTAGT ACC cag caa ctc aaa ctc gcc ttt c ** Reverse; adds SpeI site; BioSandwich compliant; no stop codons, adds a glycine.
This is the cI repressor from phage lambda tagged with a yeast codon optimized BFP.
It was important that the BFP was codon optimized for yeast.
The cI repressor was pcr amplified from a previously submitted part (BBa_K105004). The yeast codon optimized BFP was synthesized by Mr. Gene.
TPP Down-regulated Hammerhead Ribozyme 2.5 with Native RBS
It's a basic part including only a ribozyme sequence numbered 2.5
5' UTR site of alcohol oxidase 1 gene (aox1)
5' UTR site of alcohol oxidase 1 gene (AOX1) from K. pastoris used as a recombination site.
This biobrick is constructed by mutating the anticodon of tRNA（met） to TCG (base pairing codon CGA). This tRNA can transfer fMet to CGA when it is used as the start codon. Kana gene with start codon substituted for CGA is used to testify the function of metY-CGA. When this biobrick and metGM(BBa_K567014) or metG(BBa_K567015) are co-transformed into the cell, the cells can survive on the LBKana plate.
tRNA(met) has been involved in many operons, we chose metY
tRNA（met）from metY in E.coli K12
Theophylline Responsive Riboswitch P1G1 with Native RBS
Theophylline responsive riboswitch Theo P1G1
This part is the Super Yellow Fluorescent Protein 2 (SYFP2) ( BBa_K864100 ) with the strong BBa_B0034 RBS. ===Usage and Biology=== This part is usable as a reporter. The very strong fluorescence means transcription can be detected at low levels. Colonies are very clearly seen on a UV table with most promoters.
Transcriptional leakage is present in some backbones, giving visibly fluorescent colonies even without promoter.
Assembly of K864100 and the B0034 from the registry.
RBS(B0032)+T3 DNA-directed RNA polymerase
This part is the coding region of MerR, which is a mercury responsive regulator from Tn21.
We cloned this part from plasmid NR1, which was a gift from Anne O. Summers.
LasR/PAI1 Inducible Promoter + RBS (B0030) + GFP, Constitutive Promoter + RBS (B0034) + LasR
Continuous expression of LasR combined with receptor
put the constitutive promoter after the production of LasR
Mammalian-Bacterial promoter with RFP reporter: CArG promoter sequence + BBaK216005 + BBa_K08101
Our hybrid promoter hopes to add to the systems already in the registry by creating a hybrid promoter that combines the bacterial promoter PyeaR and the mammalian CArG element , in the orientation mammalian to bacterial, both of which respond to exogenous nitrogenous species. Combining the two would allow a more modular NO sensor that can be used in mammalian and bacterial cells interchangeably. The hybrid promoter has been attached to the reporter: Red Fluorescent Protein (RFP). The hybrid promoter has been characterised by observing expression of flourescent protein, and found to have increased transcription in response to increasing concentrations of potassium nitrate.
E9-ns2 CArG promoter sequence + BHN + BBaK216005 + BBa_K081014
crtY (lycopene cyclase) from Pantoea ananatis (formerly Erwinia uredovora) DSMZ 30080 (ATCC 19321). Accession: D90087. The native ribosome binding site is included. This is part of the carotenoid biosynthesis pathway, converting lycopene to beta-carotene.
This part is from previous team, but we find a point mutation in the sequence. We have correct the mutation, and this is still an available biobrick.
Pantoea ananatis (formerly Erwinia uredovora) DSMZ 30080 (ATCC 19321). Accession: D90087.
BAP2 Promoter is used to regulate the expression of genes within the genome of Saccharomyces Cerevisiae or baker's yeast.
Promoter was extracted from purified genomic DNA from Saccharomyces Cerevisiae. The PCR reaction performed also served to add the biobrick prefix and suffix to the promoter.
We constructed this part by combining R0062 and K081016. This part starts to generate LasI protein in the presence of LuxR-3OC6HSL complex.
The Ptac promoter is a functional hybrid promoter, derived from the trp and lac promoters, that are regulated by trp and lac [1]. This part also exist together with lacI, part BBa_K180000 [1] Proc. Natl. Acad. Sci. USA, Vol. 80, pp. 21-25
Oligos ordered from SigmaAldrich were annealed to form a double stranded DNA segment, ready to be ligated into any BioBrick backbone.
Created from oligos ordered from SigmaAldrich. Oligo sequences: Ptac+ AATTCGCGGCCGCTTCTAGAGGAGCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTA Ptac- CTAGTAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGATGATTAATTGTCAACAGCTCCTCTAGAAGCGGCCGCG
Medium promotor from the constitutive promotor family combined with a strong RBS and tagged with GFP to quantify the gene expression.
We made this composite to test the effiency of promotor and RBS.
LacI generator which is activated by LuxR/3OC6HSL complex
Lux Operon (under pBAD) Vibrio Fischeri
SpoVG Ribosome Binding Site (RBS) for B. subtilis
SYnthesized RBS because part BBa_K143021 was wrong.
FCY2 Promoter is used to regulate the expression of genes within the genome of Saccharomyces Cerevisiae or baker's yeast.
RhlR/PAI2 Inducible Promoter + RBS (B0030) + GFP + Constitutive Promoter + RBS (B0034) + RhlR
Continuous expression of RhlR combined with receptor with RhlP.
put the constitutive promoter after the production of RhlR
T5-lac promoter is a hybrid promoter made from the phage T5 early promoter and lac-operon. It contains three LacI binding sites and remains repressed in LacIq strains where LacI is expressed in high levels. It is inducible by IPTG, and recognizable by E.coli RNA polymerase. Compared to the T7 promoters, T5-lac promoter doesn't require the co-expression of phage polymerase.
The most common DH5alpha and TOP10 used in assembly are not LacI-constitutive (LacIq) strains. Thus, any ORF placed downstream to this promoter will be massively transcribed. Therefore, it's recommended to employ low-copy number plasmids or even LacIq strains when adding genes to this promoter.
Genomic LasR/PAI1 Inducible Promoter + RBS (B0034) + GFP, Cons. Promoter + RBS (B0030) + LasR
Genomic LasP. producing GFP along with constiutivley producing LasR
exaE is a transcription factor that is activated by phosphorylation from exaD. It acts upon pexaA.
de novo synthesis original amino acid sequence from pseudomonas aeruginosa.
Yellow Firefly Luciferase (under pBAD) L. Cruciata (E. coli optimised)
Luciferin Regenerating Enzyme (L. cruciata)
The (-)-a-pinene synthase converts geranyl pyrophosphate to 83.4%(-)-alpha-pinene, 12.6%(-)-beta-pinene, 2.1% Linalool, 1.0% (-)-beta-phellandrene, 0.4% Camphene, and 0.4% myrcene.
The (-)-a-pinene synthase (PsTPS-Pin) synthase is a monoterpene synthase isolated from Sitka spruce
SpoVG RFPmCherry double terminator. Translational unit for B. Subtilis
This is a translational unit for expressing the RFP in B. Subtilis. The RBS used is SpoVG (K143021),
This part is the fusion of the biobricks K143021 E1010 and B0015
Plasmid that consists of Acyl-ACP Reductase, rbs, normal Synechococcus elongatus PCC7942 Aldehyde Decarbonylase in PSB1C3 high consititutive vector for alkane production
Ptms+BBa_E0240: Ptms+RBS+GFP+Double
PcpcG2-B0034-BFP green light sensor output
This construct is designed to enable a quantitative analysis of the CcaS/CcaR green light-sensing system. The output is BFP ( BBa_K592100 ), which can be measured by e.g. FACS.
This part is similar to BBa_K592019 with BFP instead of amilGFP as output. While amilGFP is a good way to visualize, BFP is better for quantification.
RhlR/PAI2 Inducible Promoter + RBS (B0030) + RFP
RhlR/PAI2 inducable promoter with RBS (B0030) and RFP.
a luxR-type regulator from Rhizobium etli CFN42
raiR, a necessary part of the rai system in Rhizobium etli CFN42, can combine with the 3-OH-C8-HSL and then bind the cassette in raiI promoter, activating the expression of raiI.
The raiR is from the Rhizobium etli CFN42 from Nanjing Agriculture University.
mRFP generator controlled by PcstA (glucose-repressible promoter)
Lambda cI is controlled by promoter PcstA,which can be activated by CRP-cAMP complex.therefore protein lambda cI will be expressed under low glucose concentration.This generator contains a RFP after the promoter as a reporter.
Escherichia coli str. K-12 substr. MG1655
The sequence contains SpeI sites at positions critical for function. We modified the SpeI site in the biobrick suffix to an NheI site as done by UNAM 2010
pBAD+TPP Down-regulated Hammerhead Ribozyme 2.5 with Native RBS+E0040+B0015
This is a basic part that contains pBAD promoter, a ribozyme sequence numbered 2.5, a GFP part named E0040 and a terminator part named B0015.
Use it by stantard assembly or by sites mutation
LasR/PAI1 Inducible Promoter + RBS (B0030) + RFP, Constitutive Promoter + RBS (B0030) + LasR
Pc+ptsG(wt)-GFP (ptsG(wt) 5' UTR fused with gfp)
This is the conjugate part of the small RNA regulator sgrS_wt.
critical region for ptsG/SgrS interaction
luxEG V. Fischeri (E. coli optimised)
Red Firefly Luciferase and LRE (under pBAD) L. Cruciata (E. coli optimised)
This part generates a red-mutant of the luciferase from the Japanese firefly (L.cruciata). It also generates the luciferin regenerating enzyme. When luciferin and D-cysteine are added this BioBrick should give sustained light output. With simply luciferin it will give a burst of light output.
''Escherichia coli'' osmoregulatory trehalose synthesis B (otsB)
Rashmi Sharma, Vishesh Jain, Kendrick Wang
Modified lamdba Prm promoter (repressed by 434 cI) generating GFP. Part used for measurements.
This is a copy of the Part:B0014. Only here the part is cloned in the standard vector pSB1C3 instead of the pSB1AK3.
Modified 3' "half" of intron found in ORF #142 in Twort phage of Staphylococcus aureus in RFC53 MetaPart format. When complemented with 5' "half" BBA_K576003 in complience with BBFRFC53 assembly standard, the RNA-level excision should leave GGU linker between the whatever comes before K576003 and after K576004. K576003 and K576004 and everything in between them would be removed and possibly circularized.
The 3' end has been modified to accommodate MetaSuffix.
The RFC53 HeadPart with promoter and RBS attached.
The format is RFC53 D-HeadPart, i.e. GGT 5' overhang.
SV40NLS-GAL4AD-linker-PIF3 (part for GAL4/LexA based light-switchable promoter system)
CDS for a synthetic hybrid fusion protein. Consists of serveral domains. RFC10 pre and suffix
CDS for a synthetic hybrid fusion protein
SV40 nuclear localization sequence (recognized by importin alpha)
This is a short terminator by T7phage that we tested with our new teminators' characterization system.
This is a standard sequence, very used in many constructs. As i don't have any design considerations i can say that generally to make screening gels of such short segments is preferable to use TBE1X instead of TAE1X.
Nitrate and Nitrite sensitive promoter PyeaR with a GFP coding device and strong RBS to create a nitrate-sensitive system which signals through expression of GFP. Optimum use appears to be when sensing nitrate within a range of 0 - 20 mM
Combination of two existing biobricks from the parts registry
An RFP reporter that does not glow due to the lack of the araC gene/
The RBS, RFP, and Terminator all come from the J61002 backbone
TetR repressible promoter (BBa_R0040)+ kivd (alpha-ketoisovalerate decarboxylase)+Ter (BBa_B0015)
This is the coding sequence of kivd from Lactococcus lactis subsp. It encodes alpha-ketoisovalerate decarboxylase which is responsible for the decarboxylation into aldehydes of α-keto acids derived from amino acid transamination. This is part of the isobutanol biosynthesis pathway. This part kivd is under TetR repressible promoter that constitutively ON and repressed by TetRcan. And terminator BBa_B0015 is included.
It is cloned form Lactococcus lactis subsp. lactis KF147
Constitutive Strong promoter, Strong RBS and TAT signal peptide
Twin arginine signal peptide is an efficient protein export signal sequence of E. coli. Because of its constitutive character, its promoter works permanently.
Anderson promoter J23117 without RFP in backbone vector pSB1C3 to easily fuse the promoter with other reporters e.g. the lux operon.
Aldehyde Decarbonylase rbs Acyl-ACP Reductase FabH2 in PSB1C3 high consititutive vector for branched alkane production
RBS + bglX (E. coli perisplasmic β-glucosidase)
This is the ''E. coli'' beta-glucosidase gene ''bglX''. The part contains the native Ribosome Binding Site. The part was made using the strategy outlined in BBa_K523000 , and therefore contains 4 extra bases at the 5' end which generate a BglII restriction site.
The part was made using the strategy outlined in BBa_K523000 , and therefore contains 4 extra bases at the 5' end which generate a BglII restriction site. The part was created from a lab ''E. coli'' strain using primers: *Forward: ACT AGATCT gcc acg tcg ggc aac **adds BglII site; starts upstream of gene to catch RBS; RFC10 compliant after replacement of BBa_K523000 in a normal BioBrick vector. *Reverse: ACT ACTAGT A TTA tta cag caa ctc aaa ctc gcc **adds SpeI site; RFC10 compliant after insertion into vector; has double stop codon.
The source ''E. coli'' genome sequence can be seen using GenBank U00096.2
constitutive strong promoter with medium RBS and GFP
Strong promotor from the constitutive promotor family combined with medium RBS for strong gene expression. To quantify the gene expression, GFP was tagged to the promotor RBS domain.
We made this composite to test the effiency of the promotor and RBS domain.
Anderson promoter J23102 without RFP in backbone vector pSB1C3 to easily fuse the promoter with other reporters e.g. the lux operon.
pTet Promoter Variant #9 in Characterization Construct
This part contains a variant of the wild type Tet repressible promoter, and is followed by the pTet characterization construct, K611013. This allows for testing of this particular mutant promoter at various concentrations of repressor. The mutated pTet promoter in this plasmid expresses GFP and is followed by an arabinose inducible promoter, pBad, which expresses the TetR repressor. Thus, a user can increase the concentration of repressor by inducing pBad with arabinose. This plasmid is very useful for the further characterization of K611049.
This part does not have a termination sequence at the end. It is recommended that for optimal results, this part be used in a backbone that includes a terminator (such as pSB1C3).
Mutagenic PCR of the Tet repressible promoter, R0040, and standard BioBrick assembly of parts from the registry.
ArgR binding sites （including 8 binding sites）
Chris French, Allan Crossman, Sylvia Ispasanie
This is based on part BBa_J33207. Like that part, it encodes the lac promoter and the N-terminal 76 amino acid residues of LacZ, which produce a peptide that complements the lacZ-delta-M15 mutation in common lab strains of ''E. coli''. If grown on Xgal and IPTG, colonies with this part will be blue. The first four bases of the sequence add a BglII site (BglII sites have 6 bases, but the first 2 are provided by the standard BioBrick prefix). This enables an easy method of using PCR to create new BioBricks (e.g. from natural sources): the forward primer for the new BioBrick should have a BglII site added, and the reverse primer should have a SpeI site added. The PCR product and the plasmid containing this part can then both be digested with BglII and SpeI. After mixing and ligation, plasmids that have successfully gained the new BioBrick will be white, while those still containing the lacZ part will be blue (on Xgal+IPTG plates). One might ask: why not use the standard flanking BioBrick sites XbaI and SpeI for this procedure? The answer is that these produce compatible sticky ends and so, upon digestion, the new part will circularise.
Note that the BglII (agatct) site overlaps the BioBrick prefix. Since the prefix ends "ag", only "atct" is included in the BioBrick proper.
Based on part BBa_J33207, which was amplified from E. coli BL21 genomic DNA using primers based on published sequence (Genbank accession J01636, gi:146575).
you can insert your part behind this part so it will be immediatly expressed in e.coli.
This part conteins gvpA protein's coding sequence. This gene from Planktothrix rubescens can be succesfully expressed in E. coli. The gvpA's expression have been examined by another part " J23106+B0034+gvpA+B0015". (Since it was ligated on cloning vector pSB4A5, we didn't register this part. The gvpA protein conteins a typical domain of the superfamily 'Gas vesicle'(NCBI CDD cl02594). This gene is quiet conserved in most gvp's polycistron. According to Anthony E. Walsby's research, gas vesicles have a similar morphology. GvpA protein is a kind of small hydrophobic protein which forms the ribs of the main structure. 'All cyanobacterial gas vesicles so far analyzed contain a protein (GvpA) of about 7.4 kDa that forms the main mass of the structure and must be responsible for many of its properties.' (Gas Vesicles. Microbiological Reviews. A. E. Walsby)
The gvpA protein conteins a typical domain of the superfamily 'Gas vesicle'(NCBI CDD cl02594). This gene is quiet conserved in most gvp's polycistron. According to Anthony E. Walsby's research, gas vesicles have a similar morphology. GvpA protein is a kind of small hydrophobic protein which forms the ribs of the main structure. 'All cyanobacterial gas vesicles so far analyzed contain a protein (GvpA) of about 7.4 kDa that forms the main mass of the structure and must be responsible for many of its properties.' (Gas Vesicles. Microbiological Reviews. A. E. Walsby)
Genomic LasR/PAI1 Inducible Promoter (Short)
This is a promoter region extracted from the Pseudomonas Aeruginosa genome. It is activated by the PAI-1/LasR complex, part of the Pseudomonas quorum sensing hierarchy.
The part is unusually long for a promoter because of the primers we used to extract it (it is called short because it is shorter than companion part BBa_K575020. We do not know exactly where the regulatory region is.
It is the upstream regulatory region of [gene].
Theophyline riboswitch regulated RNA Scaffold (clover vision 2)
Scaffold D0 was constructed from a single RNA module d0, which folded into a duplex with PP7 and MS2 aptamer domains that bind PP7 and MS2 fusion proteins. A riboswitch of theophyline is added in the stucture of D0 to regulate its function.
The secquence was commercially synthesized by??????
Matthew Coyne, Ingrid Swanson, Jesa Landis, Matthew Harger
Protein originating from pseudomonas aeruginosa that confers immunity to the toxic protein Tse2
This is a human cell line optimized version of Aequorin, a bioluminescent protein found in various jellyfish and other marine organisms. The protein requires Ca2+ for catalytic activity. And oxidizes its substrate, Coelenterazine to emit blue light (λmax=469nm). Aequorin is NOT a fluorescent protein, it does not require an excitation light source to emit light. Transfection of the gene into 293T cells using lipofectamine has been confirmed. The part contains EcoRI and PstI restrictions sites, and is ligated to the iGEM pSB1C3 backbone. This backbone provides chloramphenicol resistance.
Original sequence had to be modified for increased expression in human cell lines.
Synthesized from GeneArt, original sequence from NCBI, optimized using GeneArt codon optimization software.
(correct) temperature sensitive promoter
The promoter T5-lac ( BBa_K592008 ) for testing its promoter strength coupled with BFP ( BBa_K592100 ). This construct should be cloned into pSB3K3 plasmid to measure the BFP output and thereby determine the promoter strength. Theoretically, the promoter should be well-repressed in LacIq strain but inducible by high concentrations of IPTG.
This part was assembled into pSB3K3 for testing, and later cloned into pSB1C3 for submission.
The information of BBa_K592008 and BBa_K592100 can be found in the registry. T5-lac promoter is a fusion promoter with parts from T5 phage.
Lux pL controlled LuxR with lux pR autoinducing LuxI(lva tag)- AHL.
The F2621 BioBrick device contains a lux pL regulated luxR repressor/activator and a lux pR promoter that is linked to an 3OC6HSL, an AHL autoinducer molecule, synthetase. The lux pL promoter yields a weak constitutive downstream expression. When LuxR and 3OC6HSL are produced, the lux pL promoter, however, will be repressed by a complex of the two. Complementary to the lux pL promoter, the lux pR promoter with a complex of luxR and 3OC6HSL will enable enlarged activation of the downstream synthetase gene, thus increase the concentration 3OC6HSL.
The original R0063 BioBrick part (promoter) of the F2621 BioBrick device turned out to be absent in our construct, as was shown by a sequence analysis. To increase the transcription activation there has been put an extra R0063 BioBrick part promoter in front of the F2621 BioBrick device. The resulting construct worked a lot better than the original F2621 - K082014 BioBrick system.
The lux p promoters and the autoinducer producing enzyme originate from the quorum sensing ability of ''Vibrio fischeri''.
Lux pL controlled luxR with lux pR controlled AHL degrading enzyme AiiA(lva tag).
The F2621 BioBrick device contains a lux pL regulated luxR repressor/activator and a lux pR promoter that is linked to an 3OC6HSL, an AHL autoinducer molecule, synthetase. The lux pL promoter yields a weak constitutive downstream expression. When LuxR and 3OC6HSL are produced, the lux pL promoter, however, will be repressed by a complex of the two. If 3OC6HSL is present in the cell, the autoinducer inactivation enzyme A (''aiiA''), with an LVA Tag, will inactivate the 3OC6HSL autoinducer molecules.
The original R0063 BioBrick part (promoter) of the F2621 BioBrick device turned out to be absent in our construct, as was shown by a sequence analysis. To increase the transcription activation there has been put an extra R0063 BioBrick part promoter in front of the F2621 BioBrick device.
The lux p promoters originate from the quorum sensing ability of ''Vibrio fischeri''.
a luxI homologous AHL synthetase from Sinorhizobium meliloti 1021
The Sinorhizobium meliloti strain Rm1021 harbors at least two quorum-sensing systems; one of them is sinR/sinI system, The sinI is responsible for the production of several novel AHLs, including C12-HSL, 3-Oxo-C14-HSL, C16:1-HSL, 3-Oxo-C16:1-HSL, C18-HSL, some of theses are the longest AHLs identified so far. The sin system correlates with a delay in the appearance of nitrogen-fixing nodules, as well as with an overall decrease in the number of pink nodules, suggesting a role for quorum sensing in establishing a successful symbiosis with Medicago sativa. As a autoinducer synthatase, the use of sinI is the same with luxI, namely to produce AHLS.
The Sinorhizobium meliloti 1021 is a mode organism in Rhizobia. so We got the S. meliloti 1021 from Nanjing Agriculture University easily, and then cloned the sinI gene from S. meliloti 1021 with primers designed by the part design tool in the website ginkgobioworks.com.
SinI is from the Sinorhizobium meliloti 1021. We get its gene sequence from the www.microbesonline.org, and then cloned them through PCR with biobrick part primers.
Thioesterase (TesA from E. coli) with 8-His Tag
TesA thioesterase gene from E. coli. Contains a 8-Histidine tag immediately before the stop codon to allow gel identification and extraction.
8-His tag added just before the stop codon - C terminus end of the protein. May alter activity level.
PFixK2-lambda C1-B0034-amilCP blue light sensor output
FixK2 promoter is the downstream signaling promoter of YF1/FixJ blue light-sensing system. The YF1/FixJ system become "inactive" when illuminated by blue light, and "active" in darkness. Needless to say, a system that works in the opposite way with lights activating and darkness inactivating is more desirable. Therefore, an inverter was placed behind FixK2 promoter. When in dark, YF1 becomes phosphorylated and activates FixJ, its own RR. FixJ in turn binds to FixK2 promoter, allowing the transcription of the lambda C1, thus repressing the amilCP output. In light however, YF1/FixJ has no effect on FixK2 promoter, which in turn has near-zero activity. The lambda C1 concentration drops, eventually amilCP will be produced.
The Q04510 inverter contains B0015 double terminator which gives unspecific primer binding of VR. Touchdown PCR was required to amplify the sequence for screening.
FixK2 promoter: Bradyrhizibium japonicum amilCP: Acropora millepora
It is the fusion protein of Cro and GFP with the RBS and the terminator
Enzyme in the pathway required for L-Ascorbate Acid Synthesis. This sequence was taken from plants. It catalyzes the conversion of GDP-L-Galactose to L-Galactose-1-P.
Part was constructed by ordering individual oligonucleotides. These oligos were then assembled through the use of CPEC and then further amplified with the outermost oligos as primers.
Anderson promoter J23107 (with one nucleotide different to the original Anderson promoter sequence) without RFP in backbone vector pSB1C3 to easily fuse the promoter with other reporters e.g. the lux operon.
Morgan Baltz, Katie Lutes, Akshitha Yarrabothula, Michael Wierzbicki
IRES elements initiate 5'- independent translation.
Bacterial-Mammalian Promoter: BBaK216005 + BHN + CArG promoter sequence E9-ns2
A hybrid of the PyeaR promoter (BBa_K216005) and the E9-ns2 CArG promoter (Ref:1). Two versions will be synthesised, with the promoters altering their position in relation to the 5???-end of the sequence.
To provide additional restriction enzyme sites that may become useful during later cloning steps, BamHI, HindIII and NdeI have been added between the 2 promoters. It is envisaged that any open reading frame (e.g. RFP or GFP) will be cloned ???downstream??? (i.e. at the 3???-end) of these promoter sequences.
PyeaR promoter (BBa_K216005) and the E9-ns2 CArG promoter (Ref:1) Ref1: Scott, S.D., Joiner, M.C. & Marples, B., 2002. Optimizing radiation-responsive gene promoters for radiogenetic cancer therapy. Gene therapy, 9(20), p.1396-402. Available at: http://www.ncbi.nlm.nih.gov/pubmed/12365005
promoter (lacI regulated) + a RBS (B0032)+Alss with a RBS (B0030)+ilvC
constitutive production of LasR trans-activator
Giulio Bernardinelli, Veronica Parisi, Francesca Cesaratto
iLOV has the same function and uses as LOV2. As a reporter, it is advantageous over GFP derived fluorescent proteins due to its small size (useful if you are tagging proteins), ability to recover quickly from photobleaching and use in anoxic conditions.
illegal restriction sites. The part was synthesized to remove them.
iLOV is a version of LOV2 that has been altered through site directed mutagenesis and DNA shuffling.
This is ''C. fimi'' endoglucanase under the control of the Lac promoter.
Pc+sgrS(wt)+Terminator (small RNA regulator, conjugate part of ptsG(wt))
This is the conjugate part of ptsG_wt.SgrS is the wild type small RNA regulator.
Critical region of SgrS(wt) complementary to ptsG(wt)
This chromoprotein, amilCP, naturally exhibits very strong color when expressed. The color is blue/purple and is visible to naked eye, thereby requiring no instruments to observe. This DNA was provided by Jeffrey Miller at UCLA. It was made BioBrick-compatible after removal of one illegal internal restriction site (EcoRI).
Illegal internal restriction site had to be removed (EcoRI).
Pveg is a strong, constitutive promoter of Bacillus subtilis
T7 polymerase expression casette for Bacillus Subtilis
Fused with the RBS SpoVG, this biobrick is the expression cassette for B. Subtilis of the T7 polymerase.
ccaR is the response regulator of the green sensor that also includes the membrane-associated histidine kinase ccaS. The two-componet system is also dependent of a chromophore.
The ccaR amplified from pJT118 was point mutated to get rid of illegal restriction site.
The ccaR was amplified from a pJT118 provided by Voight et al (2011) Multichromatic Control of Gene Expression in Escherichia coli, J. Mol. Biol., 405, p315???324
Upstream mutated chitoporin part fused with lacZ
ChiP encodes enterobacterial chitoporin required for uptake of chitin-derived oligosaccharides. This outer membrane porin is required for Salmonella and E.coli growth on chitooligosaccharides as a sole source of both carbon and nitrogen. It is only synthesized in the presence of these substrates. In nature it is regulated by sRNAs.In this biobrick we altered the binding site for ChiP regulator ??? ChiX. It was done in order to show that the regulatory system depends on the specific base pairing between ChiP and its regulator. The upstream part of this gene is fused with reporter gene lacZ to allow checking if the regulation works correctly.
Luminesensor repressible SulA408 promoter
SulA promoter in 408 form.SulA promoter belongs to the SOS regulon family, and promotes in E.coli transcription of SulA protein. SulA408 promoter is reponsive to our optimized Luminesensor(BBa_K819005).
SulA promoter sequence is got from regulon.org.
This part contains an RBS, the type III Zoarces elongatus antifreeze protein (ZeAFP), followed by a terminator.
Generously provided to us by Peter Davies and Sherry Gauthier of Queen's University.
Anderson promoter J23101 without RFP in backbone vector pSB1C3 to easily fuse the promoter with other reporters e.g. the lux operon.
mRFP protein generator TERM+ RBS+(B0031)
Nicolo' Politi, Federica Sampietro, Davide Bianchini
The T0 transcriptional terminator terminates transcription with 80-90% efficiency.
Contains an IPTG inducible promoter an RBS, RFP, and terminator.
A composite part containing the TetR gene, C0040, turned on by a constitutive promoter, I14033. The TetR represses the pTet promoter in front of the YFP gene. By addition of tetracycline or aTc, this repression is inhibited and the cells should fluoresce yellow.
The whole part is in the backbone of I14033. Both P0440, the TetR composite part, and E0430, the YFP composite part, were used and inserted into their respective promoters. The pTet+YFP part was then combined as a suffix insertion to the I14033+TetR backbone.
Lux pL controlled LuxR + lux pR promoter.
This part was made as a replacement for a defective version of BBa_F2621 which lacked the R0063 promoter, but was otherwise intact and functional. It shows a significantly higher response to 3OC6HSL than the incomplete BBa_F2621 (which is present in the Registry as BBa_K317037)
Was constructed by cloning BBa_R0063 in front of the incomplete BBa_F2621 (sent from the Registry in July 2011 lacking the R0063 subcomponent)
High constitutive protein expression insert includes f1 origin and RBS B0034
Jason Fontana, Daniele Rossetto, Giacomo Giacomelli
CysE is an enzyme involved In cysteine biosynthesis, also known as SAT. It catalyzes the acetylation of serine to give O-acetylserine, the CysE final precursor. Some O-acetylserine is also converted to N-acetylserine, which in turn triggers the assimilation of sulfate through specific genes. This mutated version results to be not inhibited by Cys overproduction, as it secretes Cys.
The construct has a strong RBS (AGGAGG) spaced 8nt from the start codon.
PCR extraction from E. coli genome and mutagenesis.
These cassette is encodes an autoregulated resolvase(serine type recombinase)from E.coli F plasmid transposon tn1000(tn3 family). Its promoter region consists of 3 sub-sites(res site) and can process recombination. This cassette can resolve multimer that formed during replicative transposition, so it can also resolve plasmid multimer providing plasmid stability by avoiding dimer catastrophe. For this reason, it is multimer resolution system(MRS) that provide the same function as E.coli chromosome XerCD/dif but acts independent of cell cycle and may have higher efficiency on plasmids compared with slow XerCD system.
The native sequence have two PstI site, these sites have been cleared by mutagenesis.
E.coli F plasmid transposon tn1000(tn3 family)
This is the B0010 part extracted by PCR from the doulbe terminator B0012. We didn't manage to extract it directly from the registry and we discovered that others had the same problem; for this reason we are submitting it once again, confirmed by sequencing and by experience...ready to use! The characterization has been done both with T7 and Coli RNApolymerases.
I don't have any design considerations, anyway it is useful to use TBE1X instead of TAE1X to make screening gels of such small fragments.
This part comes from B0012 contained in E0840, a double terminator in which B0010 is the second one. We used an extraction PCR.
Mun Ching Lee, Sylvia Ispasanie, Allan Crossman
''E. coli'' amylase gene ''malS'' under control of lac promoter. LacZ is also present for blue/white screening. This contains the native ''malS'' RBS.
Medium promotor from the constitutive promotor family combined with medium RBS and GFP. To quantify the gene expression, GFP was tagged to the promotor RBS domain.
We made this composite to test the effiency of the gene expression.
RhlR/PAI2 Inducible Promoter + RBS (B0030) + GFP
RhlR/PAI2 inducable promoter with RBS (B0030) and GFP.
phage shock protein A: one of the components of out Tat cassette
This parts is a coding region of phage shock protein A. It ease the difference of proton potential between periplasm and cytoplasm. The expression of this protein increase the amount of secreted proteins via Tat pathway.
This parts are derived from Escherichia coli's genomic sequence.
RhlR/PAI2 Inducible Promoter + RBS (B0034) + GFP + Constitutive Promoter + RBS (B0034) + RhlR
BBa_K607035 is a pUC18 origin of replication. [edit] Usage and Biology BBa_I50032 yields vectors with an expected copy number of 30-40 per cell.
Obtained by PCR on a plasmid prep of a pUC 18 vector.
Mammalian-Bacterial promoter with eCFP reporter: CArG promoter sequence + BBaK216005 + BBa_E0420
Our hybrid promoter hopes to add to the systems already in the registry by creating a hybrid promoter that combines the bacterial promoter PyeaR and the mammalian CArG element , in the orientation mammalian to bacterial, both of which respond to exogenous nitrogenous species. Combining the two would allow a more modular NO sensor that can be used in mammalian and bacterial cells interchangeably. The hybrid promoter has been attached to the reporter: enhanced Cyan Fluorescence Protein (eCFP). The hybrid promoter has been characterised by observing expression of flourescent protein, and found to have increased transcription in response to increasing concentrations of potassium nitrate.
This biobrick contains the RBS and terminators, meaning we can simply add the promoter of choice to complete the system.
E9-ns2 CArG promoter sequence + BHN + BBaK216005
LasR/PAI1 Inducible Promoter + RBS (B0034) + GFP
The inducable Promoter for the LasR/PAI1 complex attached to RBS (B0034) and GFP.
Need to ensure no leaky transciption of GFP.
Red Firefly Luciferase and LRE L. Cruciata (E. coli optimised)
This part is contains the coding sequence for a mutant light-emitting enzyme (luciferase) and luciferin regenerating enzyme (LRE) from the Japanese firefly, Luciola cruciata. It will make bacteria emit light at a reddish wavelength when placed under a promoter, provided the media is supplemented with luciferin. The luciferin regenerating enzyme will convert oxyluciferin into CHBT. CHBT is converted to more luciferin in the presence of D-cysteine.
Codon optimisation, making compatible with BioBrick assembly format.
This was codon-optimised for E.coli and syntesised by DNA 2.0.
YhjH protein contain a EAL domain to catalyze c-di-GMP into GMP. c-di-GMP is a magic molecule that can activates bcsB gene, a major component of cellulose synthetase complex. Also, c-di-GMP can serve as a second messager to inhibit motility and increse biofilm formation and adhesion of bacteria. Thus, the expression of YhjH gene can make the bacteria more motile.
Blue Fluorescent Protein ( BBa_K592100 ) with B0034 RBS. One can add any promoter upstream of this gene.
The BFP part ( BBa_K592100 ) was codon-optimized for expression in E.coli by DNA2.0
The coding sequence is composed from the N-Terminus to the C-Terminus by the secretion signal of human IgG in fusion with the TEM-1 beta lactamase.
This basic part has been designed in order to have a prefix and a suffix according to the standard Biobrick structure.
Synthetic origin, it has been designed according to its functions in our final system.
CysE is an enzyme involved In cysteine biosynthesis, also known as SAT. It catalyzes the acetylation of serine to give O-acetylserine, the CysE final precursor. Some O-acetylserine is also converted to N-acetylserine, which in turn triggers the assimilation of sulfate through specific genes.
LasR/PAI1 Inducible Promoter + RBS (B0030) + GFP
The inducable Promoter for the LasR/PAI1 complex attached to RBS (B0030) and GFP.
Need to ensure no leaking transcription of GFP.
PliaI promoter of Bacillus subtilis
PliaI is the promoter of the PliaI gene of Bacillus subtilis, which is inducible by bacitracin. It does not contain a ribosome binding site.
a RBS (B0034)+ilvC with a RBS (B0034)+ilvD
Marie Burghard, Henrik Cordes, Jascha Diemer, Adrian Eilingsfeld, Sven Jager, Rene Sahm, Arne Wehling
A cutinase is a cuticula degrading hydrolase from the fungus Fusarium solani pisi. It shows activity towards PET.
Surface Expression is very hard to do. FsC shows lipolytic activity and attacks the outer membrane of gramm negativ bacteria. This activity leads to a certain toxicity for expression hosts. In addition FsC needs to be expressed in the periplasm due to folding issues.
Cloned from vector pEST100 from Becker et all 2005: (Becker, S., Theile, S., Heppeler, N., Michalczyk, A., Wentzel, A., Wilhelm, S., Jaeger, K.-E., et al. (2005). A generic system for the Escherichia coli cell-surface display of lipolytic enzymes. FEBS letters, 579(5), 1177???82. doi:10.1016/j.febslet.2004.12.087)
Illegal PstI restriction site removed by silent mutations T>A at position 66 and A>T at position 69 using primers listed below (annealing sequences are underlined) ompF_rsr_F CTGAAATCTATAACAAAGATGGCAA ompF_rsr_R CTGCGTTTGCAGTACCTGCT
Amplified from MG1655 (accession NC_000913) chromosome using primers listed below (annealing sequences are underlined) ompF_BB_F TTCGAATTCGCGGCCGCTTCTAGATGATGAAGCGCAATATTC ompF_BB_R CGGCCGCTACTAGTATTATTAGAACTGGTAAACGATACC
Weiyue Ji, Hanchi Lin, Dingqiao Wen, Handuo SHI
We plan to use it in a logic gate with one input CAI-1, which can induce pqrr.
The promoter qrr is from Vibrio cholera.
Add a promoter and a RBS on the upstream will express tetR protein and GFP. GFP can help detecting tetR expression.
Genomic LasR/PAI1 Inducible Promoter (Long)
The part is unusually long for a promoter because of the primers we used to extract it. We do not know exactly where the regulatory region is.
pLux-Lac:GFP:Term:Term:PluxR/cI-OR :mCherry:Term:PtetR:LuxR:Term:Plux/cI-OR:LuxI
This is a LuxR/LacO-regulated GFP, LuxR/cI-regulated mCherry with LuxR/cI-regulated AHL amplification, TetR-regulated LuxR production.
Lipopolysaccharide (LPS), or endotoxin, is the major mediator of septic shock, a serious complication of Gram-negative bacterial infections in humans. Molecules that bind LPS and neutralize its biological effects or enhance its clearance could have important clinical applications. Limulus anti-LPS factor (LALF) binds LPS tightly, and, in animal models, reduces mortality when administered before or after LPS challenge or bacterial infection. The wedge- shaped molecule has a striking charge distribution and amphipathicity that suggest how it can insert into membranes. The binding site for LPS probably involves an extended amphipathic loop, and it has been proposed that two mammalian LPS-binding proteins will have a similar loop. The amphipathic loop structure may be used in the design of molecules with therapeutic properties against septic shock.
Optogenetical AND-Gate blue light - without first promoter - choose your first input
luxI protein generator TERM- RBS+(B0031)
Nicolo' Politi, Tommaso Goggia, Paolo Magni
Theophylline Responsive Riboswitch P1G1 with Native RBS+E0040+B0015
It is a theophylline responsive riboswitch containing an original ribosome binding site.
It can be used as a reporter of gene expression intensity.
mRFP protein generator TERM+ RBS+(B0032)
Plasmid that consists of Acyl-ACP Reductase rbs mutant Synechococcus elongatus PCC7942 Aldehyde Decarbonylase in PSB1C3 high consititutive vector for alkane production
RhlR/PAI2 Inducible Promoter + RBS (B0034) + GFP + Constitutive Promoter + RBS (B0030) + RhlR
Blue chromoprotein amilCP ( BBa_K592009 ) with B0034 RBS. One can add any promoter upstream of this gene.
amilCP was made BioBrick-compatible after removal of two illegal restriction sites.
Promoter Pveg, RBS spoVG and SacB signal peptide for B. subtilis
Constitutive promoter veg(BBa_K143012) coupled to the strong Ribosome Binding Site spoVG(BBa_K143021) from B. subtilis. These parts have been taken from 2008_Imperial_Collage. Pveg is a constitutive promoter that constitutively expresses the P43 protein in B. subtilis. SpoVG is an endogenous ribosome binding site from B. subtilis. The sequence of the spoVG ribosome binding site is AAAGGUGGUGA which is complementary to the sequence UUUCCUCCACU from the 3' region of the 16s rRNA from B. subtilis. SacB is a signal peptide used in the Sec-SRP (secretory signal recognition particle) pathway by B. subtilis. Signal peptides are responsible for directing preproteins (secretory proteins with a signal peptide region attached) through an appropriate secretory pathway. In the case of the Sec-SRP signal peptide, they direct preproteins from the cytoplasm into the growth medium.
All three basic components come from B.subtilis genome
TnsABC+D transposase machinery causes the site-specific insertion of the Tn7 transposon in the attTn7 (from attachment site) sequence, which is found in many bacteria. This insertion site is well conserved in Gram-negative bacteria, an even in higher organisms until humans. However, here we add a portable attTn7 to allow using the miniTn7 BioBrick toolkit in any organism as well as in plasmids. All the derivatives of the miniTn7 BioBrick toolkit could be inserted in the attTn7 site by transposition.
The attTn7 site was flanked by prefix and suffix for cloning purposes.
The attTn7 was obtained from the purified genome of E. coli MC4100 by PCR (using a high accuracy Pfu polymerase) with these primers: gaattcgcggccgcttctagagTGCAGCTGCTGGCTTACCATG and ctgcagcggccgctactagtaACATGGAGTTGGCAGGATGTTTG.
YF1 is a fusion protein of YtvA (B.subtilis) and FixL(Br.japonicum). It contains the N-terminal region of YtvA (1-127) and C-terminal region of FixL (258-505), interconnected by a helical linker. This fusion protein is designed by Andreas Moglich and Keith Moffat. As shown in previous research, Illumination of the fusion kinase YF1 reduced net kinase activity by ∼1000-fold in vitro. YF1 also controls gene expression in a light-dependent manner in vivo.
The sequence information was provided in Moglich and Moffat's article. The physical DNA was synthesized by GenScript. Codon-optimization may enhance its expression.
Ytva comes from the genome of Bacillus subtilis. FixL derives from the genome of Bradyrhizobium japonicum.
This is device that includes an inducible promoter, but requires the insertion of AraC to be able to induce the promoter with arabinose.
The RBS, YFP, and double terminator come from BBa_E0430.
Genomic LasR/PAI1 Inducible Promoter + RBS (B0034) + GFP
tphA3: Catalyzes together with tphA2 TPA to DCD
RhlR/PAI2 Inducible Promoter + RBS (B0034) + GFP
RhlR/PAI2 inducable promoter with RBS (B0034) and GFP.
Promoter of the liaG gene of Bacillus subtilis, weak constitutive
Yeast exportable His-rich peptide w/enhanced import (1)
This composite device produces and exports a Histidine rich peptide, that is import enhanced (thanks to a trojan peptide). The high level structure of the device is: * Kozak consensus sequence for initiation of translation * Signal peptide that targets the product of the gene for secretion * Trojan peptide, to increase internalization in target cell * Payload: this is the exported amino acid rich domain of the protein
lldPRD operon promoter + RBS from E. coli
lldP is a promoter associated with the lldPRD operon of E. coli. The lldPRD operon encodes a lactate-dehydrogenase, and the lldP promoter has been shown to be induced by L-lactate. The activity of lldP may be repressed by chloramphenicol and nutrient-rich growth media.
B0034-YF1-B0034-FixJ, blue light sensor and RR with RBS
YF1 works in conjunction with its response regulator, FixJ. This part consists of RBS-coupled YF1 and FixJ without promoter.
This constructs Strong RBS to ensure good expression.
The components that made up of YF1 fusion protein comes from Bradyrhizobium japonicum and Bacillus subtilis. FixJ comes from the genome of Bradyrhizobium japonicum.
tphB: reaction from DCD to protocatechuate
Anderson promoter J23106 without RFP in backbone vector pSB1C3 to easily fuse the promoter with other reporters e.g. the lux operon.
This is a modified version of BBa_K265008 for use with the BioSandwich protocol of Edinburgh 2011.
Tyrosine amber supressor tRNA double terminator
Tyrosine amber suppressor tRNA for B. Subtilis and double terminator.
RhlR/PAI2 Inducible Promoter + RBS (B0034) + RFP + Constitutive Promoter + RBS (B0030) + RhlR
EPIC Firefly Luciferase (under pBAD) P. Pyralis (E. coli optimised)
Plasmid that consists of Acyl-ACP Reductase, rbs, mutant Synechococcus elongatus PCC7942 Aldehyde Decarbonylase in PSB1C3 high consititutive vector for alkane production
Made by assembling [http://partsregistry.org/wiki/index.php?title=Part:BBa_I13453 BBa_I13453] with [http://partsregistry.org/Part:BBa_P0440 BBa_P0440]. Production of TetR is regulated by the pBAD promoter([http://partsregistry.org/Part:BBa_I13453 BBa_I13453]). TetR is an inhibitor of the constitutively "on" promoter pTet--[http://partsregistry.org/wiki/index.php?title=Part:BBa_R0040 BBa_R0040]). Therefore, this part may be used in conjunction with the pTet promoter as an "inverter"; pTet will be "turned off" in the presence of arabinose. Arabinose -> pBAD "on" -> TetR -> pTet "off" Intermediate part to assemble the "Co-localization Construct".
Intermediate part to assemble the "Co-localization Construct".
[http://partsregistry.org/wiki/index.php?title=Part:BBa_I13453 BBa_I13453] and [http://partsregistry.org/Part:BBa_P0440 BBa_P0440] were both obtained from iGEM distribution plates.
Cro, Lamda Repressor which activates the lytic cycle
Cro is produced by the pr promoter in the lambda phage system. Normally C1 is repressing the pr promoter. However, when the lytic switch is thrown and RecA cleaves the C1 dimer the pr promoter is unrepressed. This results in the production of cro which in turn represses the prm promoter which halts the transcription of C1 repressor. In our project we used Cro to assemble our lambda system.
Galen Lynch's notebook (Penn State iGEM 2007 member)
TPP Down-regulated Hammerhead Ribozyme 2.5 with Native RBS+E0040+B0015
Yangyang ZHAO, Yan GONG, Zhenrun ZHANG, Qingyang XIAO
This part is TPP up-regulated hammerhead ribozyme, consisting of 131 nucleotides.
This part contains a native ribosomal binding site
This part was an engineered RNA controller composed of a TPP aptamer from B.subtilis and a hammerhead motif.
pLacI Regulated Lumazine Synthase with Transciptional Terminator and pBAD
Made by assembling [http://partsregistry.org/wiki/index.php?title=Part:BBa_K542001 BBa_K542001] with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K542003 BBa_K542003]. Note that the pLacI promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010 BBa_R0010]) is regulating the Lumazine Synthase gene, not the pBAD promoter ([http://partsregistry.org/Part:BBa_I13453 BBa_I13453]) which is downstream. Therefore, Lumazine Synthase production is IPTG inducible. The microcompartment portion to the "Co-localization Construct".
The microcompartment portion to the "Co-localization Construct".
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K542001 BBa_K542001] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K542003 BBa_K542003] were both assembled by the Lethbridge 2011 team.
a luxI homologous AHL synthetase from Rhzobium etli CFN 42
RaiI is responsible for the synthesis of 3-OH-C8-HSL, a kind of AHL. The rai system in Rhzobium etli CFN42 can take the example of Rhizobium leguminosarum bv. vicae. RaiI is positively autoregulated by RaiR and 3-OH-C8-HSL but is also induced to a lesser extent by 3-OH-C14:1-HSL(produced by cinI) and 3-oxo-C8-HSL (produced by traI). These observations help explain the effect of cinRI mutations on the production of some of the short-chain AHLs and also suggest that an additional 3-oxo-C8-HSL-producing locus may contribute to regulation of raiRI.
pBAD-pXyl is a combined promoter of D-Xylose and L-arabinose sugar sensor systems which is designed to be activated only in the presence of both sugars in the medium therefore functioning as an AND logic gate. BIOLOGY pXyl is an inducible promoter regulated by the transcriptional regulator XylR which, in Bacillus subtilis, regulates the expression of xyl operon[1]. Gene expression under pXyl can be induced by the addition of D-Xylose to the medium [1, 2]. The nucleotide sequence was obtained from Wilhelm, M &C. P. Hollenberg, 1985[3]. In the presence of L-arabinose, expression from pBAD is turned on while the absence of L-arabinose produces very low levels of transcription from pBAD [4, 5]. More precisely, in the absence of arabinose, the repressor protein AraC (BBa_I13458[6]) binds to the AraI1 operator site of pBAD and the upstream operator site AraO2, blocking transcription[7], but in the presence of arabinose, AraC binds to it and changes its conformation such that it interacts with the AraI1 and AraI2 operator sites, permitting transcription[7]. The nucleotide sequence was similar of that in Part:BBa_K206000[8]. For iGEM UNAM Genomics M??xico 2012 project [9], pBAD/pXyl was used in the design of an AND logic gate[10] using a recently described new type of communication system between Bacillus Subtilis cells called Nanotubes[11].
REFERENCES [1] D Gartner, M Geissendorfer, & W Hillen(1988). Expression of the Bacillus subtilis xyl Operon Is Repressed at the Level of Transcription and Is Induced by Xylose J Bacteriol 170:7,3102-3109. [2] Shamanna, D. K., and K. E. Sanderson. 1979. Genetics and regulation of D-xylose utilization in Salmonella typhimurium LT2. J. Bacteriol. 139:71-79. [3] Wilhelm, M., and C. P. Hollenberg. 1985. Nucleotide sequence of the Bacillus subtilis xylose isomerase gene: extensive homology between the Bacillus and E. coli enzyme. Nucleic Acids Res. 13:5717-5722. [4] Lee, N. (1980) Molecular Aspects of ara Regulation. In The Operon, J. H. Miller and W. S. Reznikoff, eds. (Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory), pp. 389-410. [5] Lee, N., Francklyn, C., and Hamilton, E. P. (1987). Arabinose-Induced Binding of AraC Protein to araI2 Activates the araBAD Operon Promoter. Proc. Natl. Acad. Sci. USA 84, 8814-8818. [6] http://partsregistry.org/wiki/index.php/Part:BBa_I13458 [8] http://partsregistry.org/Part:BBa_K206000 [7] Schlief, R. (2000). Regulation of the L-arabinose operon of Escherichia coli. Trends in Genetics. 16(12):559-565. [9] http://2012.igem.org/Team:UNAM_Genomics_Mexico [10] http://2012.igem.org/Team:UNAM_Genomics_Mexico/Project/Description [11] Dubey GP, Ben-Yehuda S. (2011) Intercellular nanotubes mediate bacterial communication. Cell.;144(4):590-600 [12] Guzman, L.-M., Belin, D., Carson, M. J., and Beckwith, J. (1995). Tight Regulation, Modulation, and High-Level Expression by Vectors Containing the Arabinose PBAD Promoter. J. Bacteriol. 177, 4121-4130. [13] David, J. D., and H. Weismeyer. 1970. Control of xylose metabolism in Escherichia coli. Biochim. Biophys. Acta 201: 497-499.
Super Yellow Fluorescent Protein 2 (SYFP2)
This part codes for the bright yellow fluorescent protein SYFP2. SYFP2 is a GFP based monomeric protein with a narrow fluorescence emission spectrum with a maximum at 515 nm. Bacteria expressing SYFP2 are reported to be 12 times brighter than those expressing EYFP(Q69K) and almost 2-fold brighter than bacteria expressing Venus (Ref. 10.1021/bi0516273). Codon optimized for expression in E coli by DNA 2.0. Mutations compared to wtGFP amino acid sequence (GenBank Accession number M62653) are F46L F64L S65G S72A M153T V163A S175G T203Y A206K.
The sequence has been codon optimized for expression in E coli by DNA 2.0.
Amino acid sequence taken from the article by Kremers et al 2006.
A stop codon was added to the end of the CDS to terminate translation of sfGFP1-10
Constructed from 2011 iGEM DNA Repository Plates and Boxes, Spring Distribution, BBa_I746908
XylE - catechol 2,3-dioxygenase from P.putida with terminator
Catechol or catechol 2,3-dioxygenases + O(2) is converted by a ring cleavage into 2-hydroxymuconate semialdehyde which is the toxic and bright yellow-coloured substrate. This is a key enzyme in many (soil) bacterial species used for the degradation of aromatic compounds. The enzyme used here itself originating from Pseudomonas putida is a homotetramer of C230 monomers. The tetramerization interactions position a ferrous ion critical for enzymatic activity. It has been deduced that intersubunit interaction is essential to produce a functioning enzyme after performing N and C terminal modifications on the monomer. Coming together the subunits generate an active site. The reaction itself takes place within seconds after the addition by Pasteur pipette or spraying of catechol at a 100mM stock solution diluted with DDH20 (used by our lab.) The toxic byproduct is thought to interfere with cell wall integrity and cellular machinery such that exposed cells gradually die.
The parts were put together using standard assembly [http://partsregistry.org/Assembly:Standard_assembly]
XylE was obtained from the registry BBa_K118021 , the terminator is BBa_B0014
Mutant Regulatory protein/promoter characterization plasmid
This part can be used to characterize promoter/repressor and promoter/activator pairs by swapping in the desired parts on either end. To characterize promoter mutants, ligate the corresponding repressor or activator on the 3' end by cutting the plasmid with speI and pstI and the regulatory protein with XbaI and pstI. Then put the desired mutant promoter on the 5' end. Repressor/activator mutants can also be characterized by simply putting a wildtype promoter on the 5' end and adding the mutant repressor or activator on the 3' end. Expression is reported via GFP fluorescence. The amount of repressor/activator can be controlled using the arabinose inducible pBAD promoter. Transcriptional termination takes place within the psb1C3 plasmid which means a terminator does not need to be added after the inserted coding sequence is added.
When a protein coding sequence is added, there is no need to add a terminator since termination happens in the psb1C3 plasmid.
This part comes entirely from parts already available in the Parts Registry.
The Potsdam Assembly Standard is a modified PLICing (Phosphorothioate-based ligase-independent gene cloning) method ([http://www.ncbi.nlm.nih.gov/pubmed/20646988 Blanusa ''et al.'' (2010)]). For this standard we designed a new cloning backbone with an RFP expression cassette as insert and two new restriction enzyme recognition sites in the suffix and prefix in pSB1C3, the Potsdam Standard Backbone. In the prefix, we added the Apa I recognition site and in the suffix the Sph I recognition site. Both enzymes causing a 3??? overhang with 4 nucleotides. For the cloning process we cut the vector with Apa I and Sph I. In this assembly standard that???s the only step where we have to use restriction enzymes. The gene of interest was amplified with primers that contain 4 phosphothioate nucleotides and the recognition sites for Apa I and Sph I at the 5??? end. After incubation in an iodine/ethanol solution, the thiophosphates were cut out resulting in a 3??? overhang which is suitable to the overhangs that was created by cutting the new assembly vector. After that the digested vector and the PLICed insert were mixed and transformed into E. coli. By using the RFP expression cassette we created a ligation control system. Due to the fact that red fluorescent colonies have a failed vector ligation we can tell which colonies are correctly ligated. The colonies that do not show red fluorescence are the positive clones.
To create the Potsdam Standard Backbone, we amplified the RFP expression cassette (BBa_J04450) to add the restriction site for Apa I and Sph I. Then, the amplified expression cassette was insert into pSB1C3 using Xba I and Pst I.
This is a version of BBa_K523001 but with no RBS and no stop codon. There are two extra bases at the end which, when combined with the T at the start of the standard RFC10 suffix, code for a glycine. This part is intended to be used with Edinburgh 2011's "BioSandwich" protocol, in which a BglII site before the coding sequence, and a SpeI site after the coding sequence (i.e. the one in the suffix) are used for assembly.
The part was made using the strategy outlined in BBa_K523000 , and therefore contains 4 extra bases at the 5' end which generate a BglII restriction site. The part was created from a lab ''E. coli'' strain using primers: * ACT AGATCT atg aaa ctc gcc gcc tgt ttt c ** Forward; adds BglII site; starts at start codon. *ACT ACTAGT ACC ctg ttg ccc tgc cca gac g ** Reverse; adds SpeI site; RFC23 and BioSandwich compliant; no stop codons, adds a glycine.
tphC: terephtalate periplasmatic binding proteine of the tripartite transporter family
Valentina Herbring, Sebastian Palluk, Andreas Schmidt
tphC subunit of ABC tricarboxylate transporter together with the tctA and tctB subunits it forms the complet tricarboxylate transporter.
Wang,Y.Z., Zhou,Y. and Zylstra,G.J., Molecular analysis of isophthalate and terephthalate degradation by Comamonas testosteroni YZW-D, Environ. Health Perspect. 103 SUPPL 5, 9-12 (1995) Sasoh,M., Masai,E., Ishibashi,S., Hara,H., Kamimura,N., Miyauchi,K. and Fukuda,M., Characterization of the terephthalate degradation genes of Comamonas sp. strain E6, Appl. Environ. Microbiol. 72 (3), 1825-1832 (2006)
Reference promoter BBa_J23101 for testing the promoter strength coupled with BFP (( BBa_K592100 )). Once this is cloned into pSB3K3 backbone one can measure the BFP output and use it as reference value to measure the promoter strength of other promoters.
Also known as Glutathione S-transferase 1-1, this protein is supposed to degrade DDT, an endocrine disruptor and persistent organic pollutant.
The part was assembled through PCR using a set of long oligos. The sequence contains an NdeI site, which made it more difficult to place into a pET11a vector for protein purification and characterization.
This part was isolated from Anopheles dirus. The team took the amino acid sequence found in the NCBI protein database and used reverse transcribed using codon optimization for E. coli to create the DNA part.
pHyperspank Tyrosine amber supressor tRNA double terminator
Tyrosine amber supressor tRNA expression system induced by IPTG in presence of LacI
L-rhamnose-inducible promoter is capable of high-level recombinant protein expression in the presence of L-rhamnose, it is also tightly regulated in the absence of L-rhamnose by the addition of D-glucose.
One base pair modified (90: A -> T) to avoid EcoRI site. Mutation made in a less conserved base pair.
Amplification of the plasmid pJOE3075 (Dr. Altenbuchner).
Anderson promoter J23100 without RFP in backbone vector pSB1C3 to easily fuse the promoter with other reporters e.g. the lux operon.
It's a transcription factor for Colanic acid with a strong RBS
Melanopsin is a photosensitive protein that triggers a signalling cascade in mammalian cells.
Constitutive promoter [J23101] with mRFP, RBS B0032
Susanna Zucca, Federica Sampietro, Giuseppe Bertoni
RBS with N-terminal Oligo Arginine - ECFP Fusion
This part has a oligo arginine sequence fused to the N-terminus of an enhanced cyan fluorescent protein. This will be a component of the proof-of-concept part BBa_K331018 which will show localization of proteins into the interior of a microcompartment formed by the assemly of lumazine synthase proteins.
This sequence was synthesized by Gene Art
We chose not to include a transcriptional promoter to this part because we wanted the freedom to put it under the control of varied promoters. We also chose not to include a transcriptional terminator to this part because we planned to use it in an operon, where we would be expressing multiple proteins from a single transcript.
tctB_162: small subunit B1 of the tripartite tricarboxylate transporter family
This part was designed to test the functionality of the constitutive promotor region.
rrnB P1-LacI-pLac-mCherry plausible stress sensor
The stringent response in bacteria is caused by amino-acid starvation, fatty acid limitation, iron limitation, heat shock and other stress conditions. As a response under these conditions, ''in vitro'' studies have suggested that the alarmone guanosine tetraphosphate (ppGpp) increase to modulate transcription to promote survival. The increase in ppGpp levels causes a redirection of transcription so that genes important for survival are favoured at the expense of those required for growth and proliferation [1]. So, could we use ppGpp as signal molecule to find out when cells are stressed? '''The solution''' Our system will be based on a promoter that is important for regulating growth and proliferation. At the moment we are trying to use the ''rrnB-p1'' promoter, which has been shown in earlier studies to be highly regulated by the ppGpp molecule. Hopefully the promoter will be down regulated enough by increased levels of ppGpp to turn the repressor ''lacI'' it controls completely off. The lacI represses a second promoter ''pLac'' that induces the production of red fluorescent protein (mCherry) and turns the cells red. Reference: http://www.sciencedirect.com/science/article/pii/S0966842X05000788 1 Magnusson, L. U., A. Farewell, et al. (2005). "ppGpp: a global regulator in Escherichia coli." Trends Microbiol 13(5): 236-242
Made by combining BBa_K639002, BBa_K639000. BBa_K639002 is an improved version of BBa_K112118 made by PCR-amplification with BBa_K112118 as template. BBa_K639000 is made solely from existing registry parts.
a RBS (B0030)+ilvC with a RBS (B0030)+ilvD
Theophylline Responsive Riboswitch 1G1 with Engineered RBS
It is a theophylline responsive riboswitch.
Get this part only by PCR, rather than enzyme digestion.
Theophylline responsive riboswitch Theo 1G1
Sylvia Ispasanie, Mun Ching Lee, Eugene Fletcher
The ''E. coli'' periplasmic β-glucosidase gene bglX under the control of the lac promoter. The native ribosome binding site is present.
pBAD+Theophylline Responsive Riboswitch P1G1 with Native RBS+E0040+B0015
It is a theophylline responsive riboswitch containing an original RBS.
cI Repressible Promoter Characterization Construct
This part is used to characterize mutants of BBa_R0051 (c1 repressible promoter). By adding a mutant of BBa_R0051 before this part, one can measure several of its properties through the expression of GFP. Certain characteristics of the mutant, such as the binding of the c1 repressor, can be measured after inducing the pBad promoter with arabinose. Concentration of the repressor can be varied through different arabinose concentrations as long as AraC is present.
There is no terminator at the end, so the part needs to be used in a plasmid that has a terminator after the BioBrick suffix (such as pSB1A3 or pSB1C3). This part contains the PBad promoter without AraC, thus, a strain that already includes AraC is recommended in order to induce PBad.
Built from existing parts in the registry though BioBrick assembly.
LasR/PAI1 Inducible Promoter + RBS (B0030) + RFP
LasR/PAI1 inducable promoter with RBS (B0030) and RFP.
atgaacctcatcaaagaggatatgcgcgtaaaagtccacatggaaggcaacgtgaatggtcatgcgttcgtaatcgaagg tgaaggcaaagggaaaccgtacgaaggtacgcaaactgcaaacttgaccgtgaaagaaggtgcacctctgccattcagct acgacattcttaccactgcggttcactatggcaatcgtgtctttacgaaatatccggaagatattccggactacttcaaa cagtcgtttccggaaggctattcatgggagcgtaccatgacctttgaggataaaggcatttgcacaattcgcagcgatat tagcctggaaggggactgcttctttcagaacgttcggtttaaaggcacgaattttcctccgaatgggccggttatgcaga agaaaaccctgaaatgggaaccgtcgacagagaaactgcatgtgcgtgatggacttctggtcggtaacatcaacatggcc ttactgttggaaggtggaggccattatctctgtgacttcaaaaccacgtataaggcgaagaaagtggtacagctgccaga tgcccactttgtggatcatcgcatcgaaattctgggcaatgactccgattacaacaaagtcaagctgtacgagcatgctg ttgctcgctatagtcccttaccctctcaagtgtggtgataa
When our bacteria are growing in absence of oxygen, ZsYellow1 gets expressed. Why? FNR, is a DNA binding protein that regulates a large family of genes involved in cellular respiration and carbon metabolism during conditions of anaerobic cell growth. That is because FNR proteins are sensitive to little fluctuations in environmental oxygen concentration (FNR is believed to contain a redox/O2-sensitive element for detecting the anaerobic state). These proteins recognise a DNA target consisting of an inverted repeat: TTGATN1N2N3N4ATCAA (where N 1-4 represents a non-conserved tetrad, NCT). Then, when there is no oxygen in the media, FNR proteins bind to promoter and the transcription is activated
The sequence of the promoter comes from E. coli genome, whereas the sequence encoding the AmCyan fluorescent protein is not natural, since it was optimized for codon usage and maximum fluorescence production.
TPP Up-regulated Hammerhead Ribozyme 1.20 with Native RBS+E0040+B0015
When the bacteria suffer heat shock the protein gets expressed. Why? Heat-shock response is mediated by the Sigma 32 factor. This alternative sigma factor lets the RNA polymerase binds to some consensus promoter sequence. groE has this sequence in the promoter. That is because this gene encodes a chaperon protein, GroE. Chaperone proteins are a group of proteins present in all cells, many of them are heat shock proteins, whose function is to assist the folding of other proteins in the newly formed protein synthesis. In the case of GroE, it processes a nonnative polypeptide in a cycle consisting of three steps. First, the polypeptide substrate is captured by GroEL. Upon binding of the co-chaperone GroES and ATP, the substrate is then discharged into a unique microenvironment inside of the chaperone, which promotes proper folding. After hydrolysis of ATP, the polypeptide is released into solution. Moreover, GroE may actively increase the folding efficiency, e.g. by unfolding of misfolded protein molecules. This chaperon has an important role in heat shock proccess too, helping other proteins not to denature.
The sequence of the promoter comes from E. coli genome, whereas the sequence encoding the AsRed2 fluorescent protein is not natural, since it was optimized for codon usage and maximum fluorescence production.
PlepA is the promoter of the PlepA gene of Bacillus subtilis. It is a constitutive promoter and does not contain a ribosome binding site.
Lov-tetR (light sensor with Ptet repression) light->PtetO repressed
This part code a protein as a light sensor and repressor. With the existence of this protein, expression downstream the PtetO promoter will be repressed under the 450nm blue light. This protein, which is actually a well designed fusion protein, include two domain. The Lov domain is from Avena sativa phototrophin1(AsLOV2). The tetR is from the tetracycline resistance, which could combine the tetracycline and enable the expression of the tetA. We carefully designed the linker between these two parts, and fulfilled our sensor part in our biowave circuit.
This part is designed for our biowave project.
This coding sequence is form the Avena sativa phototrophin1(AsLOV2) and tetracycline resistance. The whole gene is synthesized by Invitrogen company.
This is the intermediate of the K914000 part, so you can change the promoter easily.
The terminator is mutated, but it does not impact the function.
A composite part containing the TetR gene, C0040, turned on by a constitutive promoter, I14033. The TetR represses the pTet promoter in front of the GFP gene. By addition of tetracycline or aTc, this repression is inhibited and the cells should fluoresce green.
The whole part is in the backbone of I14033. Both P0440, the TetR composite part, and E0240, the GFP composite part, were used and inserted into their respective promoters. The pTet+GFP part was then combined as a suffix insertion to the I14033+TetR backbone.
First enzyme in the pathway required for B-Carotene Synthesis. This enzyme is also known as Geranylgeranyl diphosphate synthase. This sequence was taken from a WT strain of xanthophyllomyces dendrorhous. It catalyzes the conversion of Fernesyl Diphosphate to Geranylgeranyl diphosphate
Part was PCR amplified from the genomic DNA created from the experiment and paper: High-Level Production of Beta-Carotene in Saccharomyces cerevisiae by Successive Transformation with Carotenogenic Genes from Xanthophyllomyces dendrorhous (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1932764/).
Found in the genomic sequence of Xanthophyllomyces Dendrorhous. http://www.ncbi.nlm.nih.gov/nuccore/AY177204.1
This part is the BBa_K864100 ( BBa_K864100 ) with the medium strength BBa_B0032 RBS. The B0032 RBS is commonly used for promoter testing, giving a wider dynamic range than a stronger RBS.
Assembly of K864100 and the B0032 from the registry.
AID is known to be responsible for somatic hypermutation and the class-switch recombination of immunoglobulin in B cells. This enzyme of 28 kDa originally occurs in B cells but does also show activity after transfection into CHO cells. AID induces the deamination of cytidine to uridine at actively transcribed single strand DNA. The replacement of cytidine by uridine leads to a mismatch during DNA replication and integrates a single base substitution predominantly in the immunoglobulin genes.
AID was amplified with primers which contains thiophosphates at 5' end and insert in Potsdam Standard Backbone using PLIcing method.
A new LL 37 bacteria toxin optimized for E. coli expression. The toxin disrupt the membrane from outside and is armless from inside.
Theophylline Responsive Riboswitch 1G1 with Engineered RBS+GFP generator
Siyang HAO, Tong MU, Zheng LUO, Mengqi ZHANG
This is a GFP generator with a theophylline responsive riboswitch sequence. The mRNA with a theophylline riboswitch in it would respond to theophylline concerntration, producing different amount of GFP. Under the control of different promoters, it could be used to demonstrate the theophylline responsive curve, providing data for modeling. The theophylline riboswitch sequence could also be used to regulate the translation of other genes.
The RBS of this device is altered into AGGAGGU. When changing the RBS, the anti-RBS sequence should be changed as well.
Theophylline riboswich is composed of a synthesized theophylline aptamer and a regulatory platform.
a RBS (B0032)+ilvC with a RBS (B0032)+ilvD
Coding region of Thiamine E from synechocystis
LasR/PAI1 Inducible Promoter + RBS (B0034) + RFP
LasR/PAI1 inducable promoter with RBS (B0034) and RFP.
The apple odor generator (BBa_K395602) catalyzes the conversion of the precursor 2-methylbutanol and butanol to the odor 2-methybutyl acetate and butyl acetate that has a apple smell. The biosynthetic device is composed of two devices:odor enzyme generator (BBa_K395601) and a T7 terminator (BBa_K395600).
T7 promoter+lac operator+RBS+His tag+thirombin+S・tag+Factor Xa+MpAAT1+His tag+T7 terminator
Promoter hyper-spank is an inducible promoter that has been designed for high expression in B.subtilis. Gene expression under the control of the promoter hyper-spank can be induced by addition of Isopropyl β-D-1-thiogalactopyranoside (IPTG).
This leader sequence originally from the 5'-untranslated RNA of the rpoS gene. Its particular secondary structure places the ribosome binding site into a double-stranded region and therefore prevents recognition by the ribosome. A small RNA, called dsrA can interacts with it and induces a change that liberates the RBS and thus stimulates the translation the downstream gene.
It has been extracted from colony by PCR and cloned into PSB1C3.
lexA3 is lexA (BBa_K864201) with a G85D mutation (G>A basepair change at position 254)
Anderson promoter J23118 without RFP in backbone vector pSB1C3 to easily fuse the promoter with other reporters e.g. the lux operon.
Lac promoter driving expression of crtEIB cluster.
This is the lac promoter driving Enhanced Yellow Fluorescent Protein. It was made as a test of the BioSandwich protocol of Edinburgh 2011.
BioSandwich Assembly of K523000 and K523012
Orange Firefly Luciferase (under pBAD) L. Cruciata (E. coli optimised)
EPIC Firefly Luciferase and LRE P. Pyralis (E. coli optimised)
This is a mutant luciferase that is very much brighter than wild-type. It is supplied in an operon with an enzyme (LRE) that regenerates its substrate. When supplied with luciferin and placed under a promoter it will emit light at a green wavelength. If D-cysteine is added the LRE will be able to regenerate the luciferin substrate.
It was ensured that no unwanted restriction sites were created during optimisation. A BamH1 site was added between the two genes.
Codon optimised and synthesised by DNA 2.0
Luciferin regenerating enzyme (P.pyralis)
RBS(B0034)_MBP(lead metal binding peptide egineered from PbrR)+Terminator(B0015)
This part is combined with a rbs, a mbp which codes for the metal binding peptide and a terminator.
Bas Stringer (Sequence by Paul van Dieken)
This part encodes the protein ''cochaperonin 10'', which is part of the Cpn10/Cpn60 chaperonin system of ''Oleispira antarctica''. [More information will follow soon.]
The protein's sequence[1] was published by Ferrer ''et al.'' in 2004. Pre- and suffixes were added to this as clarified in OpenWetWare's BioBrick standards[2] and, as is recommended, the TAA stop codon was replaced with TAATAA. The sequence's conformity with Assembly standard 10[3] was confirmed using the EMBOSS recoder[4], by recoding restriction sites of EcoRI, XbaI, SpeI, PstI, NotI, PvuII, XhoI, AvrII, NheI and SapI. 1 - http://www.ncbi.nlm.nih.gov/nuccore/22266159?from=458&to=751&report=gbwithparts 2 - http://openwetware.org/wiki/Biobrick_standard 3 - http://partsregistry.org/Help:Assembly_standard_10 4 - http://bioweb2.pasteur.fr/docs/EMBOSS/recoder.html
Ptms - a constitutive promoter in E. coli and B. subtilis.
Mammalian-Bacterial Promoter: E9-ns2 CArG promoter sequence + BHN + BBaK216005
A hybrid of the PyeaR promoter (BBaK216005) and the E9-ns2 CArG promoter (Ref1). Two versions will be synthesised, with the promoters altering their position in relation to the 5???-end of the sequence.
PyeaR promoter (BBaK216005) and the E9-ns2 CArG promoter (Ref1) Ref1: Scott, S.D., Joiner, M.C. & Marples, B., 2002. Optimizing radiation-responsive gene promoters for radiogenetic cancer therapy. Gene therapy, 9(20), p.1396-402. Available at: http://www.ncbi.nlm.nih.gov/pubmed/12365005 [Accessed June 24, 2012].
Promoter veg a constitutive promoter for B. subtilis
Pveg is a constitutive promoter that constitutively expresses the P43 protein in ''B.subtilis''. Pveg contains binding sites for the ''B.sutbilis'' major sigma factor#1. Pveg in ''B.subtilis'' utilises two binding sites to cause high expression of genes#2, however our Pveg is lacking the upstream site to give a medium level of gene expression. It has been noted that the sporulation master regulatoion factor spoOA interacts with Pveg though it is not known how#3. The context with which we used the promoter Pveg is as a '''Polymerase Per Second''' (PoPS) generator.
The biobrick part was designed to include a single binding site for the ''B.subtilis major sigma factor. In addition the biobrick standard was applied to the promoter Pveg sequence.
The Pveg promoter was suggested to us by Dr. Jan-Willem Veening of Newcastle University. This sequence supplied was compared to that of the DBTBS database#3 then a section containing the binding site synthesised by Geneart.
reflectin for b.subtilis with LipA signal peptide
Constitutive promoter veg(BBa_K143012) coupled to the strong Ribosome Binding Site spoVG(BBa_K143021) from B. subtilis. These parts have been taken from 2008_Imperial_Collage. Pveg is a constitutive promoter that constitutively expresses the P43 protein in B. subtilis. SpoVG is an endogenous ribosome binding site from B. subtilis. The sequence of the spoVG ribosome binding site is AAAGGUGGUGA which is complementary to the sequence UUUCCUCCACU from the 3' region of the 16s rRNA from B. subtilis. LipA is a signal peptide from the B. subtilis genome. In general, signal peptides are responsible for directing preproteins (secretory proteins with a signal peptide region attached)through an appropriate secretory pathway[3]. LipA has been successfully used in the secretion of heterologous proteins such as cutinase by B. subtilis. Reflectin is a self-assembling protein that has the ability to reflect the sun light which depends on the thickness of the protein layer. Therefore, we thought that this ability could be used as a novel reporter for B.subtilis and E.coli.
RBS with N-terminal Oligo Arginine - EYFP Fusion
This part has a oligo arginine sequence fused to the N-terminus of an enhanced yellow fluorescent protein. This will be a component of the proof-of-concept part K331018 which will show localization of proteins into the interior of a microcompartment formed by the assemly of lumazine synthase proteins.
pBAD+TPP Up-regulated Hammerhead Ribozyme 1.20 with Native RBS+E0040+B0015
This is a basic part that contains a promoter, a ribozyme sequence numbered 1.20, a GFP part named E0040, a terminator part named B0015.
TPP Up-regulate Hammerhead Ribozyme 1.20
RBS + malS (E. coli periplasmic α-amylase)
This is the ''E. coli'' Amylase gene ''malS''. The part contains the native Ribosome Binding Site. The part was made using the strategy outlined in BBa_K523000 , and therefore contains 4 extra bases at the 5' end which generate a BglII restriction site.
The part was made using the strategy outlined in BBa_K523000 , and therefore contains 4 extra bases at the 5' end which generate a BglII restriction site. The part was created from a lab ''E. coli'' strain using primers: *Forward: ACT AGATCT gaa atc gca gca ata agg act c **adds BglII site; starts upstream of gene to catch RBS; RFC10 compliant after replacement of BBa_K523000 in a normal BioBrick vector. *Reverse: ACT ACTAGT A TTA tta ctg ttg ccc tgc cca g **adds SpeI site; RFC10 compliant after insertion into vector; has double stop codon.
The expected sequence can be seen using GenBank U00096.2
Anderson promoter J23103 without RFP in backbone vector pSB1C3 to easily fuse the promoter with other reporters e.g. the lux operon.
Promoter can be induced to express a marker gene (GFP) when exposed to galactose in the absence of glucose.
This promoter was taken from the S. cerevisiae Advanced Gateway Destination Vectors.
Acyl-ACP Reductase in PSB1C3 high consititutive vector
This part is used to characterize mutants of BBa_R0040 (pTet). By adding a mutant of BBa_R0040 before this part, one can measure several of its properties through the expression of GFP. Certain characteristics of the mutant, such as the binding of the TetR repressor, can be measured after inducing the pBad promoter with arabinose. Concentration of the repressor can be varied through different arabinose concentrations as long as AraC is present.
HHO1 yeast UTR is used to regulate the expression of genes within the genome of Saccharomyces Cerevisiae or baker's yeast.
UTR was extracted from purified genomic DNA from Saccharomyces Cerevisiae. The PCR reaction performed also served to add the biobrick prefix and suffix to the UTR.
3' UTR site of alcohol oxidase 1 gene (aox1)
3' UTR site of alcohol oxidase 1 gene (AOX1) from K. pastoris used as a recombination site.
The DNA sequence from plasmid pPIC9K of Invitrogen (http://products.invitrogen.com/ivgn/product/V17520?ICID==%3Dsearch-product) was used for primer design. But the genomic DNA of Pichia pastoris wildtype X33 was used for amplification via PCR.
CsgD, the master regulator of biofilm formation
Promoter Pveg, RBS spoVG and LipA signal peptide for B. subtilis
Constitutive promoter veg(BBa_K143012) coupled to the strong Ribosome Binding Site spoVG(BBa_K143021) from B. subtilis. These parts have been taken from 2008_Imperial_Collage. Pveg is a constitutive promoter that constitutively expresses the P43 protein in B. subtilis. SpoVG is an endogenous ribosome binding site from B. subtilis. The sequence of the spoVG ribosome binding site is AAAGGUGGUGA which is complementary to the sequence UUUCCUCCACU from the 3' region of the 16s rRNA from B. subtilis. LipA is a signal peptide from the B. subtilis genome. In general, signal peptides are responsible for directing preproteins (secretory proteins with a signal peptide region attached)through an appropriate secretory pathway[3]. LipA has been successfully used in the secretion of heterologous proteins such as cutinase by B. subtilis.
Tse2/Tsi2, toxin antitoxin system. Tse2: A toxic protein originating from pseudomonas aeruginosa that has been shown to arrest growth in both prokaryotic and eukaryotic cells when expressed intracelluarly. It is a substrate p. aruginosas type 6 secretion system. Tsi2: Protein originating from pseudomonas aeruginosa that confers immunity to the toxic protein Tse2.
Rcn is a promoter that is activated by cobalt. A repressor is fixed to the promoter by default, and cobalt binds to this repressor, which activates the gene under control of this promoter. Rcn is also activated by nickel, but we have not characterized this aspect.
An internal iGEM restriction site had to be mutated by QuickChange mutagenesis.
Anderson promoter J23114 without RFP in backbone vector pSB1C3 to easily fuse the promoter with other reporters e.g. the lux operon.
A composite part containing the TetR gene, C0040, turned on by a constitutive promoter, I14033. The TetR represses the pTet promoter in front of the RFP gene. By addition of tetracycline or aTc, this repression is inhibited and the cells should fluoresce red.
The whole part is in the backbone of I14033. P0440, the TetR composite part was used and inserted into its respective promoter. The RBS+RFP+Terminator part came from the backbone J61102 and was inserted into the pTet promoter. The pTet+RFP part was then combined as a suffix insertion to the I14033+TetR backbone.
Green Firefly Luciferase and LRE (under pBAD) L. Cruciata (E. coli optimised)
This is the lux operon from V. fischeri expressed under a pBAD promoter. It emits light when induced by arabinose. luxCDE create a substrate which the luciferase complex of luxA and luxB uses to emit light at a blue wavelength.
Removal of existing lux promoter, and replacement with pBAD
From pHK555 provided by James Slock ( http://departments.kings.edu/biology/lux/bacterial.html )
Medium promotor from the constitutive promotor family combined with a weak RBS and tagged with GFP to quantify the gene expression
The (-)-b-pinene synthase (PsTPS-Pin2) synthase is a monoterpene synthase isolated from Sitka spruce converts geranyl pyrophosphate to 70.5%(-)-beta-pinene, 29.5%(-)-alpha-pinene.
The (-)-b-pinene synthase (PsTPS-Pin2) synthase is a monoterpene synthase isolated from Sitka spruce.
Mating factor alpha 1 with Kozak sequence
The Kozak sequence is based on the BioBrick BBa_J63003, but the sequence is used without the last bases atggag. And the MFalpha1 sequence is from the plasmid pPICZalpha A from Invitrogen.
This part is a heat shock promoter, it refers to the transcription factor sigma32. when the temperature rise, the transcription factor sigma32 expresses a lot and binds to the RNA ploymerase to recognize the conserved sequence in -35 and -10 region, then the promoter starts to transcript.
The whole sequence of this part is only 90bp, so take a futher consideration if you intend to digest it for use.
We align the HSP promoters from the literature and then find the conserved sequence, then we just modified the promoter of groES of E.coli, and synthesis the part through BIC genescript.
Promoter for constitutively high expression.
CARIM, Sean; LAM Ka Shing; MOK Ka Chun; SHI, Tianxing
Evan Clark, Julius Ho, Eli Moss, Jesse Palmer
Anabaena vegetative cell constitutive promoter. Can be used to express specifically in vegetative cells, excluding heterocyst expression.
pHyperspank Tyrosine amber supressor tRNA
RhlR/PAI2 Inducible Promoter + RBS (B0030) + GFP + Constitutive Promoter + RBS (B0030) + RhlR
This is a coding part comprising the full open reading frame of Salmonella enterica LT2 hcp gene (also known as STM0289 or tssD) linked via an haemagglutinin eptitope tag to a synthetic gene encoding an endolysin from the CD27 bacteriophage. The CD27 endolysin is believed to have high specificity against the cell wall of Clostridium difficile.
The hcp section is derived from BBa_K895001 , which was used as a PCR template, and the endolysin section is derived from a plasmid constructed by Neil Fairweather's group at Imperial College London.
LacI Repressor and Promoter Wild Type Control
This part is can be used as a control for BBa_K611010 and BBa_K611013, which test either C0012 and R0010 mutants respectively.
IAA biosynthetic genes under control of the Pveg2 promoter
The construct of IaaM and IaaH genes in PVEG plasmid
This is GFP from Biobrick BBa_J54103 , which we have sequenced and re-cloned to RFC[10] standard in pSB1C3.
Express GFP only if protein CI activated the promoter pRM.
Fusion part of pBAD arabinose-inducible induction system and the HRV 14_3C protease
FixK2 promoter is the wild-type promoter to which phospho-FixJ binds. FixJ in turn can be regulated by YF1, the blue light-sensing protein. This promoter shows very little leaky activity in the absence of FixJ.
The sequence information was provided by Moffat (Moffat et al. 2009). The DNA was synthesized by GenScript.
FixK2 promoter omes from the genome of Bradyrhizobium japonicum.
This part codes for the bright blue fluorescent protein mTagBFP. mTagBFP is a monomeric protein with a narrow fluorescence emission spectrum with a maximum at 456 nm. It has a tyrosine-based chromophore, giving it substantially higher brightness, faster chromophore maturation and higher pH stability than blue fluorescent proteins with a histidine in the chromophore. Subach et. al. started with a red fluorescent protein (TagRed) and converted it to a bright blue monomeric protein. See the article listed in source. The sequence has been codon optimized for expression in E coli by DNA 2.0.
Subach, O. M., I. S. Gundorov, et al. (2008). "Conversion of red fluorescent protein into a bright blue probe." Chem Biol 15(10): 1116-24.
A B.subtilis promoter regulated by sigma G late in Sporulation in the forespore
It is the centromere site that can be recognized by parB of pseudomonas putida KT2440, when providing this part in cis on an low copy plasmid and providing parAB cassette in cis or in trans, the plasmid can be active partitioned thus it can enhances plasmid segregation stability and reduces plasmid loss rate.
It had better be used in low copy plasmid.
LasR/PAI1 Inducible Promoter + RBS (B0030) + GFP, Constitutive Promoter + RBS (B0030) + LasR
This logical gate is based on amber stop-codon suppression via the non-canonical tRNA supD. The blue light sensor induces the expression of a mRNA coding for a T7-polymerase with an amber mutation, that can only be translated by ribosomes if the correct supD tRNA is present. The supD tRNA is expressed when the red light sensor is induced. Thus, only cells which receive both signals produce the desired substances.
We used YcgF/E as blue light sensor and Cph8 as red light sensor. We expect that this assembly will be the one with the lowest amount of unspecific gene expression which could occur due to overlapping of the absorption spectrums of the sensory domains.
The particularity of lox71 is that it has an altered sequence at the end of it's right arm compared to loxP. This sequence variation reduces affinity of the Cre recombinase for the arm.
Based on the report made by the igem2010 UT-Tokyo team and some papers, the lox71 (BBa_I718017) from the registry was wrong, it had a cg instead a gc in its center sequence that is between the arms. Apparently, this part was corrected by the iGEM11_WITS_CSIR_SA team (BBa_K537020), but the sequence from this group had the same error. The corrected part from iGEM11_Tokyo_Tech (BBa_K649205) was right but no DNA was available in the registry. So we decided to synthesized it and test it, using the proper sequence described by http://www.ncbi.nlm.nih.gov/pmc/articles/PMC137435/, and we submitted the correct DNA to the Registry.
a luxI homologous AHL synthetase from Rhzobium leguminosarum bv. vicae IMA57
R. leguminosarum bv. viciae is a best characterized organism in the nitrogen-fixing rhizobia. Several quorum-sensing systems (rai, rhi, cin,and tra) have been identified and are intertwined in a complex regulatory network. The rhi system is composed of rhiR (a luxR homolog),rhiI (a luxI homolog), and the rhiABC operon, all of which are located on the symbiotic plasmid pRL1JI (29, 139). It was demonstrated that rhiABC was controlled by RhiR and that flavonoids repressed the expression of both rhiR and rhiABC.
Initial experiments showed 3-OH-C14:1-HSL to be an inducer of the rhi genes, but later data showing that rhiI and rhiA could be induced by short-chain AHLs (C6-HSL, C8-HSL, and 3-oxo-C8-HSL) but not 3-OH-C14:1-HSL suggested that the induction was indirect.
it is from the Rhzobium leguminosarum bv. vicae IMA57 we purchased from the CGMCC(China General Microbiological Culture Collection Center).
KRE9 yeast UTR is used to regulate the expression of genes within the genome of Saccharomyces Cerevisiae or baker's yeast.
GFP reporter regulated by 3OC12-HSL and LasR.
Phage origin recognized by M13. Used to make SS DNA when infected with M13k07 helper phage
This part is an RFP coding device. When ligated with a promoter, it will yield a red colony so that you can determine whether your promoter works.
LacI Repressor Characterization Construct
This part is used to characterize mutants of BBa_C0012 (LacI). By adding a mutant of BBa_C0012 after this part, one can measure several of its properties through its regulation of GFP expression. Concentration of the added TetR mutant can be varied through different arabinose concentrations as long as AraC is present.
There is no terminator at the end, so the part needs to be used in a plasmid that has a terminator after the BioBrick suffix (such as pSB1A3 or pSB1C3).
lambda cI generator controlled by PcstA (glucose-repressible promoter)
Lambda cI is controlled by promoter PcstA,which can be activated by CRP-cAMP complex.Therefore protein lambda cI will be expressed under low glucose concentration.
This reporter part is a combination of a constitutive promoter, AraC gene, a pBad promoter (inducible with arabinose), and a GFP gene. The whole part is flanked by the regular E, X and S, P restriction sites, so it can be approached with the usual restriction digest method. The purpose of this part is to provide the user with a simple, but complete, inducible genetic device. Adding arabinose with any concentration higher than 0.01 mM seems to cause a significant expression of the GFP gene. This is useful if the user plans to build a larger plasmid construct in a short time or characterize a specific part. This part can be inserted in a cell line that does not contain any AraC in its genome, which is important if the user is not using Top 10 cells.
There is no specific design consideration.
This part is obtained from ligating several biobricks.
B.subtilis transformation vector with LacI
This part is designed for integration into ''B. subtilis' genome at amyE locus, it contains Spectinomycin resistance selection marker and constitutively expresses LacI. PmeI can be used to ligate a gene of interest using blunt ended methods.
Existing biobricks, BBa_K143070 , BBa_K316002 , BBa_K316014 BBa_K143002
''Deinococcus radiodurans'' Superoxide dismutase, SodCu/Zn
The RFC 53 TailPart with transcription terminator.
The format is RFC53 D-TailPart, i.e. ATG 5' overhang.
This part is used to characterize mutants of BBa_R0010 (pLacI). By adding a mutant of BBa_R0010 before this part, one can measure several of its properties through the expression of GFP. Certain characteristics of the mutant, such as the binding of the LacI repressor, can be measured after inducing the pBad promoter with arabinose. Concentration of the repressor can be varied through different arabinose concentrations as long as AraC is present.
Reflectin is a self-assembling protein that has the ability to reflect the sun light which depends on the thickness of the protein layer. Therefore, we thought that this ability could be used as a novel reporter for B.subtilis and E.coli.
Optimized for both B.subtilis and E.coli
Medium constitutive protein expression insert includes f1 origin and RBS B0034
kivd (alpha-ketoisovalerate decarboxylase) under TetR repressible promoter
This is the coding sequence of kivd from Lactococcus lactis subsp. It encodes alpha-ketoisovalerate decarboxylase which is responsible for the decarboxylation into aldehydes of α-keto acids derived from amino acid transamination. This is part of the isobutanol biosynthesis pathway. This part kivd is under TetR repressible promoter that constitutively ON and repressed by TetR.
kivd is cloned form Lactococcus lactis subsp. lactis KF147
Tyrosine amber suppressor tRNA for B. Subtilis.
This sequence is the pure tRNA without the side sequences
Synthetize de novo by matching primers and cloned from the tRNA cluster of B. Subtilis genome of strain 168
TDH3 Promoter is used to regulate the expression of genes within the genome of Saccharomyces Cerevisiae or baker's yeast.
promoter (lacI regulated)+RBS+Alss with a RBS (B0032)+ilvC+a RBS (B0032)+ilvD
Promoter region from E.coli containing the binding site for the global regulator Crp.
Victor Alvarez, Felix Reyes and Yolanda Gonzalez
Proteolysis is a very common system to regulate gene expression in all organisms. In bacteria, proteins can be targeted for proteolysis by adding a special degradation tag in their coding sequences. That???s how the Sspb-Clpx system works. In E.coli wild type, ClpX recognize specific degradation tags ??? ssrA tags -, unfolds the attached protein and translocates this denatured polypeptide into ClpP for its degradation, which is very fast. The action of this ClpXP protease is enhanced by the SspB adaptator protein. The idea is to add the DAS+4 tag to the proteins of one of the states of the flip-flop and express the Sspb under the promoter of the opposed state. Specifically, it's added the DAS+4 tag to the GFP and the LacI proteins, expressed under the cI promoter, and under the Lac promoter it's expressed Sspb. Furthermore, asRNAs are short non-coding RNAs that can inhibit mRNA translation by binding to the 5??? untranslated region (UTR) of that mRNA. That is the case of the Salmonella ompN gene, whose translation is inhibited by the conserved RybB asRNA, which forms a short duplex with the ompN CDS. This device has a fusion of OmpN start sequence with those proteins whose translation we want to regulate. The RybB asRNA binds to nucleotides from 5th to 20th relative to AUG. Consequently, a shorter fusion in which it's fused the first 7 codons of the OmpN gene CDS upstream of the RFP and Sspb CDS. '''These fusions do include the OmpN RBS'''. http://2011.igem.org/wiki/images/1/1a/UPOSevilla_finalconstruction.png Here we show the final structure of our improved bistable. As well as the two operons with their protein fusions and the promoter with the asRNA, we show the different restriction sites we have introduced and the enzymes that target each site. Finally, the total length of our construction is indicated in the figure. '''IMPORTANT''': This biobrick is '''incomplete''', you will need the asRNA device. You can find it in ... '''For further information please vistit our [http://2011.igem.org/Team:UPO-Sevilla wiki] or contact with us at igem@upo.es'''
Lac induced protein expression insert includes f1 origin, Lac I gene and RBS
LasR/PAI1 Inducible Promoter + RBS (B0034) + RFP, Constitutive Promoter + RBS (B0034) + LasR
This is the red light sensor Cph8 with RBS.
Since Cph8 was considerably longer than the RBS added, sequencing was necessary to confirm the construct.
The cph8 was taken from Tabor's pJT122 plasmid.
only the plasmid contain rep gene can be used in magnetic bacteria AMB-1
DNA ligase from Escherichia coli (LigA) with PT7, RBS and His-tag
Anderson promoter J23113 without RFP in backbone vector pSB1C3 to easily fuse the promoter with other reporters e.g. the lux operon.
ldhA promoter + RBS from Corynebacterium glutamicum
Promoter region associated with the ldhA operon of C. glutamicum.
Anderson promoter J23115 without RFP in backbone vector pSB1C3 to easily fuse the promoter with other reporters e.g. the lux operon.
Hampus Elofsson, Sabri Jamal, Lisa Branzell
The natural smallRNA spot42 taken from the E-coli MG1655. SmallRNA is belived to interact in many metabolic pathways. The spot42 scaffold interacts with HFQ-proteins. It is in many cases shown to bind into the rbs area of the mRNA and thereby blocking translation initiation. ( V.Sharma, A.Yamamura Y.Yokobayashi, 2011 ) We wanted to use this sRNA for gene downregulation on translational level, in this case block the ribosome from binding to the rbs area. You can use this natural scaffold to make your own sRNA, inhibiting translation on a gene of your choice. The sRNA is coupled to a strong promotor, J23101
It can be a bit tricky to do mutagenesis on it, like we did. We wanted to add a a random antisense area with primers. You cannot have any scars between the promotor and sRNA so we added our promotor with mutagenesis.
The part was synthesized by Bio Basic Inc. into the pET28a plasmid vector with an N-terminal His-tag. The Enhanced Lumazine Synthase (ELS) without the His-tag was moved into the pSB1C3 plasmid vector by the Lethbridge 2011 team. The size of the cavity of the Lumazine Synthase microcompartment was enlarged via directed evolution (W??rsd??rfer ''et al.,'' 2011). The compartment formed by this polypeptide has a larger cavity than the Lumazine Synthase microcompartment submitted by the Lethbridge 2009 team ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K249002 BBa_K249002]). A larger interior allows for the option of localizing larger and/or more proteins into the cavity of the compartment. Reference: W??rsd??rfer, B., Woycechowsky, K.J., and Hilvert, D., (2011). Directed Evolution of a Protein Container. ''Science.'' 331: 589-592.
The compartment formed by this polypeptide has a larger cavity than the Lumazine Synthase microcompartment submitted by the Lethbridge 2009 team ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K249002 BBa_K249002]).
The Enhanced Lumazine Synthase (ELS) sequence was the result of directed evolution. The part was synthesized by Bio Basic Inc. and moved into pSB1C3 by the Lethbridge 2011 team. W??rsd??rfer, B., Woycechowsky, K.J., and Hilvert, D., (2011). Directed Evolution of a Protein Container. ''Science.'' 331: 589-592.
cheZ ligated to a medium promoter. This part express CheZ protein mediumly and will induce medium mobility in delta cheZ E.coli strain.
There is a point mutation at position 56 of CheZ coding sequence with have little effect to its function and the bases corresponding to stop codon is changed to bases coding a more frequently used stop codon.
The promoter used here is from Partsregistry. The CheZ gene is from E.coli strain MG1655 by colony PCR and aligned with CheZ sequence at EcoGene database.
vgb promoter is induced under microaerobic conditions.
We construct this promoter, according to the reference: "Construction of highly efficient E. coli expression systems containing low oxygen induced promoter and partition region" by Liu Tao et al.
vgb promoter comes from Vitreoscilla sp.
Bacterial-Mammalian promoter with eCFP reporter: BBaK216005 + CArG promoter sequence + BBa_E0420
Our hybrid promoter hopes to add to the systems already in the registry by creating a hybrid promoter that combines the bacterial promoter PyeaR and the mammalian CArG element , both of which respond to exogenous nitrogenous species. Combining the two would allow a more modular NO sensor that can be used in mammalian and bacterial cells interchangeably. The hybrid promoter has been attached to the reporter: enhanced Cyan Fluorescence Protein (eCFP). The hybrid promoter has been characterised by observing expression of flourescent protein, and found to have increased transcription in response to increasing concentrations of potassium nitrate.
The enhanced Cyan Fluorescence Protein (eCFP) was chosen with the eventual aim of attaching it to part BBa_K774002 or BBa_K774003. It was therefore important that the fluorescence peak of eCFP didn't overlap with RFP too much, as eCFP and RFP will be reporting on either part BBa_K774002 or BBa_K774003 at the same time.
BBaK216005 + BHN + CArG promoter sequence E9-ns2 + BBa_E0420
The constitutive promoter region (97 bp) from the Escherichia coli K-12 tatABCD (twin-arginine translocase) operon. This is a constitutive promoter that gives good levels of expression in E. coli, but is not inducible or repressible. The promoter region is cloned as an EcoRI / PstI fragment into pSB1C3 following Biobrick Standard [10]. This is a corrected part that replaces the BBa_K562000 biobrick.
The part has an engineered BamHI (5'-GGATCC-3') restriction site at the extreme 3' end. For best results, open reading frames should be cloned into this BamHI site with their ATG initiation codons immediately following it. This will place the ATG at exactly the correct distance from the native tatA RBS for optimal translational efficiency.
SmtA is a metallothionein that can bind to heavy metal ions such as Cd(II). This SmtA is cloned from Synechococcus sp. PCC7942 and it has been reported that the cyanobacterial strains that express SmtA can resist medium containing cadmim.
This part was cloned from Synechococcus sp. PCC7942, using primers to add EcoRI, XbaI and SpeI at the ends.
Intermediate synthetic part of optogenetical AND-Gate
Cloning of part was difficult due to small fragment sizes. Therefore whole part was synthesized.
Truncated lysostaphin coding sequence. Lysostaphin has has a specific lytic action against S.aureus.
Lysostaphin is a zinc metalloenzyme that has a specific lytic action against S.aureus. Lysostaphin has activities of three enzymes namely, endo-β-N-acetyl glucosamidase, N-acteyl-muramyl-L-alanine amidase, glycylglycine endopeptidase. Glycylglycine endopeptidase lyses staphylococcal cells by hydrolyzing glycylglycine bonds in the poly-glycine bridges which form cross links between glycopeptide chains in cell wall peptidoglycan of S.aureus cells. The wild-type lysostaphin gene encodes a preproenzyme, and the conversion of prolysostaphin to mature lysostaphin occurs extracellularly and involves the removal of the hydrophilic tandem repeat portion of the proenzyme. In order to directly produce mature lysostaphin, we truncate the preprolysostaphin and prolysostaphin sequence.
In order to directly produce mature lysostaphin, we truncate the preprolysostaphin and prolysostaphin sequence.
This part was isolated from Tn21. When regulated by its cognate transcription factor MerR, it will represent a dose-response manner in response to the concentration of mercury.
AiiA protein generator TERM+ RBS+(B0032)
Susanna Zucca, Tommaso Goggia, Paolo Magni
promoter (lacI regulated)+RNA thermometer
mRFP protein generator TERM+ RBS+(B0030)
RhlR/PAI2 Inducible Promoter + RBS (B0034) + RFP
RhlR/PAI2 inducable promoter with RBS (B0034) and RFP.
This is a tetracycline responsive repressor that is tagged with yeast codon optimized BFP.
Needed the BFP to be codon optimized for yeast
BFP was synthesized Tetr is from the Registry of Parts: BBa_K319046
Nostoc punctiforme PCC73102 Aldehyde Decarbonylase in PSB1C3 high consititutive vector
LasR/PAI1 Inducible Promoter + RBS (B0030) + RFP, Constitutive Promoter + RBS (B0034) + LasR
Fusion of Ice Nucleation Protein (INP) and Enhanced Yellow Fluorescent Protein (EYFP) under the control of the Lac promoter.
Made using the BioSandwich protocol of Edinburgh 2011.
Assembled from parts BBa_K523000 , BBa_K523008 , and BBa_K523012 .
A composite part comprising of a J23100 promotor and PA2652 translational unit encoding extracellular chemoreceptor
optimized for both e.coli and b.subtilis
Colicin E2 Immunity protein under pLac promoter (BBa_R0011) with repressilator RBS. This part can be used to confer immunity against the colicin E2 activity protein by induction with IPTG.
We selected the repressilator RBS and pLac to drive production of Colicin E2 Immunity protein
hycAp: Promoter of hyc operon activated by FhlA
hycAp is promoter activated by FhlA (transcriptional activater)
LasR/PAI1 Inducible Promoter + RBS (B0034) + GFP, Constitutive Promoter + RBS (B0034) + LasR
Blue Fluorescent Protein with B0032 RBS, this part can be added downstream to any promoter and then tested in FACS.
RhlR/PAI2 Inducible Promoter + RBS (B0034) + RFP + Constitutive Promoter + RBS (B0034) + RhlR
p(Caif) facilitates the analysis of the transcriptional activity of the circuit responsive to Crp which is produced in the absence of glucose.
Nucleotide sequence optimized for expression in E.coli