Part consists of the signal peptide of Erbb1/EGF-receptor, which ensures the transport to the cell membrane scFV-antiNIP, GGGSflexlinker (extracellular parts), the trans-membrane region of the Erbb1/EGF-receptor and the N-terminal part of the split-fluorophor YFP(intracellular part).
Derived by cloning of the parts BBa_K157001, BBa_K157003, BBa_K157013, BBa_K157002, BBa_K157008. For further information see the description of the parts mentioned above.
This chromoprotein from the coral Anemonia sulcata, asCP, naturally exhibits color when expressed.
Synthesized and codon optimized for E coli by GenScript.
fwYellow, yellow chromoprotein reporter system (Strong Promoter, Strong RBS)
Team Uppsala 2012 chromoprotein attracts many interests because it's more convenient than fluorescent protein for it can be observed with naked eyes. However, characterized data of these proteins are few. Because a single coding region is on the plasmid, we constructed BBa_K1943000 with chromoprotein fwYellow(BBa_K1033910) this year and did characterization. We've constructed a series of plasmids which are all in the same pattern: a strong/weak promoter, a strong/weak RBS and a chromoprotein. We want to monitor the speed of expression of these plasmids in normal incubation conditions (like 37℃ overnight for LB agar plate and 37℃ 180rpm for LB broth). The expression speed and strength of the constructed biobricks will be carefully monitored and gave others a relative scale for using chromoprotein as a reporter gene.
This chromoprotein from the coral Montipora efflorescens, meffBlue, naturally exhibits strong color when expressed.
This part has a strong constitutive promoter (J23100) the standard RBS (B0034) and AmilCP (K592009). When assembled into a plasmid it gives a strong blue color. The reason we created this part is the default backones all have essentially the same construct but using RFP instead. With an insert with a different color it made it much easier to debug problems with digests, ligations and transformations. We also used it to try to find a problem with some strange background issues we had. This was part also use to create a lot of AmilCP that was used in our protein purification experiments.
We needed a strong blue color to make it easy to distinguish this from a red carry through. We used a strong anderson promotor (J23100), standard RBS and hooked it to AmilCP.
J23100, K592009 and B0034 came from the registry.
Produces a purple pigment (violacein) from L-tyrosine. The operon contains five genes (VioA-E) each with their own ribosome binding sites.
We added complementary restriction sites on either side of vioC and vioD (BamHI, BglII, HindIII). This allows them to be seperately removed to produce more colours from the operon.
The VioA-E genes were originally from from Chromobacterium voilaceum ATCC 12472 in the pPSX vio+ plasmid. This was kindly provided by John Pemberton; Department of Microbiology and parasitology, University of Queensland, Brisbane, Australia. (Sarovich & Pemberton (2007) Plasmid 57:306-313). The codons were optimised for E. coli by DNA2.0 (Welch, Villalobos, Gustafsson and Minshull (2009) J. R. Soc. Interface)
plasmid from Jansen lab Tuebingen University
fwYellow, yellow chromoprotein reporter system (medium Promoter , weak RBS)
Team Uppsala 2012 chromoprotein attracts many interests because it's more convenient than fluorescent protein for it can be observed with naked eyes. However, characterized data of these proteins are few. Because a single coding region is on the plasmid, we constructed BBa_K1943001 with chromoprotein fwYellow(BBa_K1033910) this year and did characterization. We've constructed a series of plasmids which are all in the same pattern: a strong/weak promoter, a strong/weak RBS and a chromoprotein. We want to monitor the speed of expression of these plasmids in normal incubation conditions (like 37℃ overnight for LB agar plate and 37℃ 180rpm for LB broth). The expression speed and strength of the constructed biobricks will be carefully monitored and gave others a relative scale for using chromoprotein as a reporter gene.
Part consists of the signal peptide of Erbb1/EGF-receptor, scFV-antiNIP, GGGSflexlinker (extracellular part), the trans-membrane region of the Erbb1/EGF-receptor and the C-terminal part of the split fluorophor YFP with a fluolinker joined to its N-Terminus (intracellular part).
Derived by cloning of the parts BBa_K157001, BBa_K157003, BBa_K157013, BBa_K157002, BBa_K157006. For further information see the description of the parts mentioned above.
lacZ (encoding beta-galactosidase, full-length)
LacZ encodes beta-galactosidase, an intracellular enzyme that cleaves the disaccharide lactose into glucose and galactose. Beta-galactosidase can also cleaves X-gal and ONPG into colorful products.
PCR amplified and standardized from BL21
Produces dark brown pigment output. The gene (melA) codes for a tyrosinase which produces the pigment from L-tyrosine. Production of the pigment requires the addition of copper and L-tyrosine supplements (the copper acts as a cofactor for the gene product) but no other precursors. The BioBrick sequence includes the native ribosome binding site.
The only changes to the sequence from the plasmid kindly provided by The Massachusetts Institute of Technology and Gregory Stephanopoulos was the removal of two resitrction sites, and the addition of the iGEM prefix and suffix. The two PstI restriction sites were removed by converting CAG to CAA in the second codon of the restriction site.
The melA gene comes from the Rhizobium etli CFN 42 symbiotic plasmid p42d (accession number U80928). The origonal plasmid was created by Christine Nicole S. Santos and Gregory Stephanopoulos (reference: http://aem.asm.org/cgi/content/abstract/74/4/1190)
Beta-galactosidase (EC 3.2.1.23) is a classic enzyme most commonly used in blue-white colony screening when used in specialized vectors. This part consists of the full length beta-galactosidase sequence which can be inserted downstream of a promoter to produce the tetrameric, 47.4kDa enzyme. Beta-galactosidase cleaves the beta-glycosidic bond of a galactose bound to an organic moiety; the physiological function is to break down beta-galactosides, such as lactose, into it's respective monnosaccharides. Beta-galactosidase can act as a reporter when provided substrates such as X-gal. The cleavage of X-gal releases galactose and the chromophoric, subtsituted indole moiety. The most common form of X-gal is 5-Bromo-3-indolyl β-D-galactopyranoside, which produces an intense blue pigment. Other versions of X-gal may be used to produce other color pigments. Typically 40-60uL of a 20ug/mL X-gal solution, dissolved in dimethyl sulfoxide or dimethyl formamide, is spread on a plate and allowed to dry before plating the bacteria expressing beta-galactosidase. X-gal may also be added to liquid cultures or incorporated into solid agar media.
The addition of BioBrick prefix and suffix.
Amplified and isolated from an expression vector.
Blue-Yellow fluorescent protein toggle switch using pLac and OmpC promoter.
Ryan Kawalerski, Sarah Heacox, Jonathan Krog
GFP - LacZ protein complex with flag tags
This part contains a green fluorescent protein (GFP) as a non-reactive chemical compound (anchor) in order to neutralize the active reactive fixed to it, a reporter device (LacZ enzyme catalyses the reaction of the cleavage of X-Gal which results with blue color change) and a TEV protease cleavage site between them to assemble these two proteins. BBa_772001 and BBa_K772002 are assembled to be used as the trigger of detection in the whole system which ends with the intracellular transfection (or activation) of TEV protease. After the activation, it is planned that TEV protease will cleave this anchor-inducer protein complex to release free the reporter. In conclusion, in this system, the inducer would activate a transcription pathway on the targeted inducible gene or the reporter would indicate that there is a change in the whole system. This part contains flag tags extra from BBa_K772006 in the GFP region; because it is planned to be used in the Co-IP/SDS-PAGE assay to be isolated.
Cleavage site, LacZ and GFP sequences are gathered from Registry.
Note that there is an RBS sequence just before the protein sequence in order to eliminate the need of addition of it.
amilCP, blue/purple chromoprotein (incl RBS)
This chromoprotein from the coral Acropora millepora, amilCP, naturally exhibits strong color when expressed. The protein has an absorbance maximum at 588 nm giving it a blue/purple color visible to the naked eye, thereby requiring no instruments to observe. The strong color is readily observed in both LB or agar culture, in less than 24 hours of incubation.
Illegal internal restriction site had to be removed (EcoRI).
Compatible with the Plug'n'Play with DNA assembly standard
Part consists of the signal peptide of Erbb1/EGF-receptor, scFV-antiNIP, GGGSflexlinker (extracellular part), the trans-membrane region of the Erbb1/EGF-receptor and the C-terminal part of the split enzyme beta-Lactamase (intracellular part).
Derived by cloning of the parts BBa_K157001, BBa_K157004, BBa_K157013, BBa_K157002 and BBa_I757012. For further information see the description of the parts mentioned above.
J23110-B0034-aeBlue (chromoprotein) (E.coli optimized)
aeBlue is a blue chromoprotein extracted from the basal disk of a beadlet anemone Actinia equina. The protein has an absorption maximum at 597nm and a deep blue colour. The protein aeBlue is of the GFP family with significant sequence similarities. This part is a functionally active variant of BBa_K864401.
The promoter J23110 is sufficiently strong to yield expression levels making the blue protein clearly visible and eventually opaque.
The protein was discovered, extracted and analyzed in (Shkrob, 2005). It was synthesized by Bioneer Corporation, codon optimized for E.coli.
Blue chromoprotein amilCP (<partinfo>BBa_K592009</partinfo>) with B0034 RBS. One can add any promoter upstream of this gene.
amilCP was made BioBrick-compatible after removal of two illegal restriction sites.
LacZ alpha fragment; complements matching N-terminal deletion mutant (lacZ-omega)
to greatly reduce the number of bases, this is only a portion of the LacZ gene
Restriction sites (modified) 76-79 (gcc to gca) 79-81 (gct to gca) 85-87 (gaa to gag) 106-108 (tgc to tgt) 109-111(agg to aga)
amajLime, yellow-green chromoprotein (incl RBS & J23110)
This chromoprotein from the coral Anemonia majano, amajLime, naturally exhibits strong color when expressed.
A composite part consisting of the signal-peptide of Erbb1/EGF-receptor, scFV-antiNIP, GGGSflexlinker (extracellular part), the trans-membrane region of the Erbb1/EGF-receptor and the N-terminal part of split-enzyme beta-Lactamase (intracellular part).
Derived by cloning of the parts BBa_K157001, BBa_K157003, BBa_K157013, BBa_K157002 and BBa_I757011. For further information see the description of the parts mentioned above.
Part consists of the signal peptide of Erbb1/EGF-receptor, scFV-antiNIP, GGGSflexlinker (extracellular part), the trans-membrane region of the Erbb1/EGF-receptor and the N-terminal part of the split enzyme beta-Lactamase (intracellular part).
Derived by cloning of the parts BBa_K157001, BBa_K157004, BBa_K157013, BBa_K157002 and BBa_I757011. For further information see the description of the parts mentioned above.
GFP - LacZ protein generator is generated with the aim of inducing a reporting reaction aftermath of the activation of EpCAM detection system. (BBa_K772100) For more information: BBa_K772006
To achieve a constitutive production, pTetR promoter is added in the upstream region of the original part.
fwYellow, yellow chromoprotein (incl RBS, J23106)
This chromoprotein naturally exhibits strong color when expressed.
Cloned from FezziwigYFP vector, DNA 2.0.
amilGFP, yellow chromoprotein (incl RBS)
This chromoprotein from the coral Acropora millepora, amilGFP, naturally exhibits strong yellow color when expressed. The color is readily visible to naked eye both in LB-culture and on agar plates. Color development can be seen in less than 24 hours of incubation.
Two illegal internal restriction sites (EcoRI and PstI) were removed.
Acropora millepora. The DNA was provided by Jeffrey Miller at UCLA, and was made BioBrick-compatible by removing two illegal internal restriction sites (EcoRI and PstI).
GFP - LacZ anchor-inducer protein complex
This part contains a green fluorescent protein (GFP) as a non-reactive chemical compound (anchor) in order to neutralize the active reactive fixed to it, a reporter/inducer device or protein (enzyme etc.) and a TEV protease cleavage site between them to assemble these two proteins. BBa_772001 and BBa_K772002 are assembled to be used as the trigger of detection in the whole system which ends with the intracellular transfection (or activation) of TEV protease. After the activation, it is planned that TEV protease will cleave this anchor-inducer protein complex to release free the inducer/reporter. In conclusion, the inducer would activate a transcription pathway on the targeted inducible gene or the reporter would indicate that there is a change in the whole system.
tsPurple, purple chromoprotein (incl RBS, J23110)
Cloned from TinselPurple vector, DNA 2.0.
aeBlue is a blue chromoprotein extracted from the basal disk of a beadlet anemone Actinia equina. The protein has an absorption maximum at 597nm and a deep blue colour. The protein aeBlue has significant sequence homologies with proteins in the GFP family.
Synthesized and codon optimized for E. coli by Bioneer Corp.
In strains with lacZ-omega (lacZ N-terminal deletion mutant) like DH5alpha, DH10B and Top10, lacZ-alpha restores the beta-galactosidase activity.
PCR amplified from BBa_J33202 without the RBS.
Alkaline phosphatase coding sequence (no rbs, promoter, or terminator present) from Citrobacter. This bacterium's phoA was chosen due simply to the absense of Biobrick restriction sites.
pre-pcr ca1033F/ca1032R on Citrobacter gen (1500 bp) PCR ca1032F/R on above PCR (1500 bp, XbaI/EcoRI) Sub into pSB1A2-Bca1008 (XbaI/MfeI, 2077+935, large) Product is pSB1A2-Bca1032 ---- ca1032F Forward oligo to biobrick phoA of Citrobacter GGTTAtctagaTGTTTTTTATTTTAACTGTATTTATACATGGAG ca1032R Reverse oligo to biobrick phoA of Citrobacter cagtaGAATTCccagaggcggcaTTACTTCAG ca1033F Forward constant oligo to clone phoA ttatcatcgctccgcatcctg
meffBlue, blue chromoprotein (incl RBS, J23110)
A composite part consisting of the signal peptide of Erbb1/EGF-receptor, scFV-antiNIP, GGGSflexlinker (extracellular part), the trans-membrane region of the Erbb1/EGF-receptor and the C-terminal part of the split-enzyme beta-Lactamase (intracellular part).
Derived by cloning of the parts BBa_K157001, BBa_K157003, BBa_K157013, BBa_K157002 and BBa_I757012. For further information see the description of the parts mentioned above.
This construct is synthetic. It was synthesized from a template where the consensus sequence for the "color dependancy" in order to obtain a chromoprotein with a unique yellow color.
Carbon Monoxide sensor device (pCooF-Blue Chromoprotein)
This part is the carbon monoxide (CO) sensing module of the 'Inspector NOxCO' device. 'Inspector NOxCO' is a bacterial sensor for carbon monoxide (CO) and oxides of nitrogen (NOx) in vehicle emissions. This part consists of the Anderson constitutive promoter(BBa_J23110) and RBS(B0030) with the coding sequence for CooA(BBa_K352001) a CO dependent transcriptional activator and single terminator(B0010) followed by CooA activated strong promoter pCooF(K352002) with RBS(B0030) and 'amilCP, blue chromoprotein' with terminator(BBa_B0012). If CO is present, the constitutive expressed CooA will bind to it and then bind to the pCOoF promoter activating transcription of the blue chromoprotein.
The selection of the RBS and Constitutive promoter. Because of limitations of repeats that could be synthesized, we had to give up on the double terminators in the original design and go with single different terminators. A 70bp spacer(B0040) has been added for transcriptional efficiency.
All parts in this sequence come from the Registry and have been listed. However, the complete part has been synthesized from IDT using their free gBLocks offer.
This is the lacZa subunit used to complement cells for blue/white screening. It has been cloned into a vector including the prefix/suffix required for standard 25 assembly for fusion proteins.
This part contains the AgeI and NgoMIV sites as part of it's prefix/suffix that allows it to be used in standard 25 assembly. It was synthesized through PCR oligo synthesis methods using the following primers: Forward TATTGAATTCGCGGCCGCTTCTAGATGGCCGGCACCATGATTACGGATTCAC (57.74 oC) Reverse ATAACTGCAGCGGCCGCTACTAGTATTAACCGGTTCACTCCAGCCAGC (55.05 oC)
This part was amplified out of registry stock of LacZa in order to include the standard 25 prefix/suffix
gfasPurple, purple chromoprotein (incl RBS)
This chromoprotein from the coral Galaxea fascicularis, gfasPurple, naturally exhibits strong color when expressed.
This part is a reporter sfGFP(Sp) originally optimized for Streptococcus pneumoniae.
sfGFP originally optimized for streptococcus pneumonia because it could be shown that the signal in Bacillus is even brighter than the one from sfGFP(Bs).
First fragment of beta-Lactamase fused to the trans-membrane region of the Erbb1/EGF-receptor.
Derived by cloning of the parts BBa_K157002, BBa_I757011. For further information see the description of the parts mentioned above.
NOx(Oxides of Nitrogen) sensor device (pNirK-Yellow chromoprotein)
This part is the oxides of nitrogen (NOx) sensing module of the 'Inspector NOxCO' device. 'Inspector NOxCO' is a bacterial sensor for carbon monoxide (CO) and oxides of nitrogen (NOx) in vehicle emissions. This part consists of the Anderson constitutive promoter(BBa_J23110) and RBS(B0030) with the coding sequence for NsrR(K2056001) a NOx sensitive repressor protein and single terminator(B0010) followed by NsrR repressed promoter pNirk(k2056002) with RBS(B0030) and 'Yellow chromoprotein'(K1033910) with terminator(BBa_B0012). If NOx are present, they will bind to the constitutive expressed NsrR that will relieve repression on the pNirK promoter activating transcription of the yellow chromoprotein.
amajLime, yellow-green chromoprotein (incl RBS)
fwYellow, yellow chromoprotein (incl RBS)
Contains an IPTG inducible promoter an RBS, RFP, and terminator.
tsPurple, purple chromoprotein (incl RBS, J23106)
This is a mutated version of the Xyle reporter gene which encodes for the enzyme catechol-2,3-dioxygenase (metapyrocatechase), which converts catechol to the bright yellow product 2-hydroxy-cis,cis-muconic semialdehyde. This version of Xyle has been made compatible with standard 25 assembly methods by removing three restriction sites (two NgoMIV sites: at bp 315 and 486, as well as one AgeI site: at bp 837). These mutations were made synonymous with the original sequence and codon optimized for E. Coli. Because of the synonymous mutations, this gene can be easily used to create fusion protein parts. For more information on the Xyle gene and its uses as a reporter see part BBa_J33204.
The mutations in this gene were made so that they were synonymous and codon optimized for E. coli cells. The part also contains the prefix/suffix for standard 25 assembly with addition restriction sites NgoMIV and AgeI.
The original genetic material for this part came from part BBa_K316007 which was mutated via PCR based site-directed mutagensis. The xyle portion of this large part was then amplified via PCR reaction and cloned into a new vector along with a prefix/suffix for standard 25 assembly.
Second fragment of beta-Lactamase fused to the trans-membrane region of the Erbb1/EGF-receptor.
Derived by cloning of the parts BBa_K157002, BBa_I757012. For further information see the description of the parts mentioned above.