araC-pBAD + β-carotene + J23100 + blh
AraC-pBAD promoter comes from E.coli genome, while ctrEIBY are from Pantoea ananatis (formerly Erwinia uredovora) genome. Blh gene and its promoter are synthetized and codon optimezed by GeneScript.
This biobrick is obtained by the assembly of two parts K1604020 and K1604021. The composite part contains the five genes necessary for the retinal synthesis. The first part contains ctrEIBY under araC-pBAD, these four gene are responsive of beta-carotene production starting from FPP, a colurless precurse presents in E.coli. The second part contains blh under costitutive promoter. It encodes for the beta-carotene15-15'dioxygenase that catalizes the cleavege of a single molecule of beta-carotene into two molecules of retinal.
The two parts was joined together by strandard assembly, before it beta-carotene was amplified by Phusion PCR.
This Composite Biobrick is created by standard assembly of Part BBa_K731201 and Part BBa_K274200, which are submitted by previous iGEM teams. The whole cassette is on plasmid pSB1C3 (high copy, Chloramphenicol resistance). BBa_K274200 contains the enzymes that convert colourless farnesyl pyrophosphate to orange beta-carotene. The carotene operon contains CrtE, CrtB, CrtI and CrtY each one with individual ribosome binding site. This Composite Biobrick has been put under inducible araC-pBAD promoter that is positively regulated by arabinose.
Standard assembly between BBA_K731201 and a PCR amplification of BBa_K274200. BBa_K274200 was extracted by the 2012 distribution kit in pSB1A2 and it was amplified by PCR to restore a missing SpEI site.
J23100 + β-carotene 15,15’-dioxygenase (blh gene)
The sequence of uncultered cluster bacteria SAR86 was synthetized and codon optimized by GeneScript
blh encodes for β-carotene 15,15'-dioxygenase, the enzyme that cleaves one molecule of β-carotene into two molecules of retinal
The part was subcloned from pUC57 into pSB1C3
This E. coli promoter is positively regulated by arabinose. araC is the regulator protein, binding to arabinose prevents the repression of pBAD.
This part is similar to BBa_I0500, but it differs from that one in several point mutations in the sequence.
RBS + β-carotene 15,15'-dioxygenase (blh gene)
Gene coding sequences come from Pantoea ananatis (formerly Erwinia uredovora) (Accession number D90087), a type of Gram negative Enterobacteriaceae. This Composite Biobrick is created by standard assembly of Part BBa_K118014, Part BBa_K118006, Part BBa_K118005 and Part BBa_K118013.
This Composite Biobrick is created by standard assembly of Part BBa_K118014 (CrtE with rbs), Part BBa_K118006 (CrtB with rbs), Part BBa_K118005 (CrtI with rbs) and Part BBa_K118013 (CrtY with rbs), which are submitted by previous iGEM teams. The whole cassette is on plasmid pSB1A2 (high copy, Ampicillin resistance). Together, enzymes CrtE, CrtB, CrtI and CrtY convert colourless farnesyl pyrophosphate to orange beta-carotene (via intermediates geranylgeranyl pyroiphosphate, phytoene and lycopene). The orange beta-carotene pigment can then be used as a coloured signal output, e.g. for biosensors. There is already individual ribosome binding site before each enzyme gene sequence. Internal restriction sites have been removed by previous iGEM teams. Please refer to Parts BBa_K118014, BBa_K118006, BBa_K118005 and BBa_K118013 for more information on individual Biobricks components. This Composite Biobrick has been put under constitutive promoter R0011 (see Part BBa_K274210) and arabinose-inducible promoter I0500 (see Part BBa_274220), transformed and tested in E.coli strain MG1655. Amount of beta-carotene produced can be measured by photospectrometer with absorbance at 455nm (beta-carotene extraction using acetone). A related Composite Biobrick is Part BBa_K274100 (CrtEBI with rbs), which "stops" at one step before beta-carotene, producing (and retaining) red lycopene pigment.
The colourless precursor, farnesyl pyrophosphate, is naturally produced in E.coli using Non-mevalonate Pathway (from pyruvate and glyceraldehyde-3-phosphate). Our experiences showed that this Biobrick works better in E.coli strains with up-regulated level of precursors. E.coli strain MG1655 demonostrates higher level of pigment (beta-carotene) production than strain TOP10.
strong synthetic E. coli ribosome binding site with SacI site.
This is a strong synthetic E. coli ribosome binding site. It is synthesised as two complementary oligonucleotides rather than being incorporated into a biobrick plasmid. It incorporates a SacI site overlapping the XbaI site, which is preserved when it is added to any other biobrick. This allows easy detection of the RBS after it has been added upstream of a biobrick coding sequence in a plasmid vector.
Note the presence of a SacI site overlapping the XbaI site, which is preserved when this biobrick is added to any other biobrick. At the time of writing, this biobrick is added as a short piece of DNA composed of two complementary oligonucleotides rather than being incorporated into a biobrick cloning vector by itself. It can be added upstream of a biobrick coding sequence, and its presence can easily be detected in miniprep DNA on a gel by using a SacI-SpeI or similar digest.
crtE (geranylgeranyl pyrophosphate synthase) coding sequence.
Pantoea ananatis (formerly Erwinia uredovora) DSMZ 30080 (ATCC 19321). Accession: D90087.
crtE (geranylgeranyl pyrophosphate synthase) from Pantoea ananatis (formerly Erwinia uredovora) DSMZ 30080 (ATCC 19321). Accession: D90087. Part of the carotenoid biosynthesis parthway.
crtB coding sequence encoding phytoene synthase
This is the coding sequence of crtB from Pantoea ananatis (formerly Erwinia uredovora) (Accession number D90087). It encodes phytoene synthase, part of the carotenoid biosynthesis pathway, which converts geranylgeranyl diphosphate to phytoene. Reference: Misawa,N., Nakagawa,M., Kobayashi,K., Yamano,S., Izawa,Y., Nakamura,K. and Harashima,K. Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products expressed in Escherichia coli. J. Bacteriol. 172 (12), 6704-6712 (1990)
crtI coding sequence encoding phytoene dehydrogenase
This is the coding sequence of crtI from Pantoea ananatis (formerly Erwinia uredovora) (Accession number D90087). It encodes phytoene dehydrogenase, part of the carotenoid biosynthesis pathway, which converts Phytoene to Lycopene.
The native crtI sequence contains two internal PstI sites. Primers crtImutf1 and crtImutf2 were ordered so that the MABEL protocol (invented for this purpose) could be used to remove these sites. The mutagenic primers were as follows: * crtImutf1: gtt tac agt aag gtt gcc * crtImutf2: agt aac tct ctg ttt gtg c In both cases, the PstI site CTGCAG was converted to CTGCAA, a silent mutation.
Synthetic ribosome binding site added by Son of Babel procedure
This is a 4bp synthetic ribosome binding site which can be added by fusion PCR using a reverse primer and the Son of Babel cloning system.
The part can be added by fusion PCR using a Son of Babel reverse primer.
crtY coding sequence encoding lycopene B-cyclase
This is the coding sequence of crtY from Pantoea ananatis (formerly Erwinia uredovora) (Accession number D90087). It encodes lycopene B-cyclase, part of the carotenoid biosynthesis pathway, which converts lycopene to B-carotene.
Conversion of the iGEM parts registry to SBOL2.1