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)
crtE (geranylgeranyl pyrophosphate synthase) with native rbs.
crtE (geranylgeranyl pyrophosphate synthase) from Pantoea ananatis (formerly Erwinia uredovora) DSMZ 30080 (ATCC 19321). Accession: D90087. The native ribosome bindind site is included. This gene is part of the carotenoid biosynthesis pathway.
This part was made as a single biobrick rather than separate rbs and coding sequence, hence the use of blunt ligation in generating the final sequence.
Pantoea ananatis (formerly Erwinia uredovora) DSMZ 30080 (ATCC 19321). Accession: D90087.
Farnesyl-Pyrophosphate-Synthase from Yeast
This is the Farnesyl-Pyrophosphate-Synthase from ''Saccharomyces cerevisiae'' which produces Farnesyl-Pyrophosphate from Isoprenyl Pyrophosphate and Dimethylallyl Pyrophosphate. It is encoded by the gen ERG20. We inserted some calm mutations to avoid conflicts with the Cloning Standard 10.
We inserted some calm mutations to avoid conflicts with the Cloning Standard 10.
The source of this part is ''Saccharomyces cerevisiae''. We amplified the sequence from genomic DNA of CEN-PK-2 using the following PCR primers:
This is the crtB gene from Erwinia uredovora with a ribosome binding site in front. This codes for the enzyme phytoene synthase and is the second step in beta-carotene production.
We got this from the plasmid pAC-3.10.10.A sent to us by Ethan Johnson of iGEM Minnesota team timebomb.
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 was made as a single biobrick rather than separate rbs and coding sequence, hence the use of blunt ends in generating the final sequence.
crtZ (beta-carotene hydroxylase) with native rbs
crtZ (beta-carotene hydroxylase) from Pantoea ananatis (formerly Erwinia uredovora) DSMZ 30080 (ATCC 19321). Accession: D90087. Part of the carotenoid biosynthesis pathway. Converts beta-carotene to the yellow pigment zeaxnathin.
This part was made as a single biobrick rather than separate rbs and coding sequence, hence the use of blunt combination to generate the final sequence.
This E. Coli enzyme (isopentenyl diphosphate isomerase or idi) is catalyzes the reaction from IPP (or DMAPP) to OPP, a precursor for the synthesis of FPP (farnesyl diphosphate).
rbs+crtE (geranylgeranyl pyrophosphate synthase)
This is the coding sequence of ''crtE'' from ''Pantoea ananatis'' (formerly ''Erwinia uredovora'') (Accession number D90087). It encodes geranylgeranyl pyrophosphate synthase, part of the carotenoid biosynthesis pathway, which converts farnesyl diphosphate to geranylgeranyl diphosphate.
The ''crtE'' BioBrick (BBa_I742151) had to be transferred from the non-compliant BABEL vector to a compliant pSB1A2 vector.
The synthetic ribosome binding site was added by fusion PCR to the ''crtE'' gene, which was amplified from ''Pantoea ananatis'' DSMZ 30080 (ATCC 19321) genomic DNA.
rbs+dxs (1-deoxyxylulose-5-phosphate synthase)
This is the coding sequence of dxs from Escherichia coli JM109 with added ribosome binding site (rbs). It encodes 1-deoxyxylulose-5-phosphate synthase, thiamine-requiring, which catalyses the first step in the biosynthesis of terpenoids. Overexpression of dxs has been reported to increase yields of carotenoids and other terpenoids [add reference here].
The last enzyme required for FPP synthesis. A geranyltranstransferase from E. Coli (ACB01549).
This part consists of a lac promoter (with lacZ' tag for blue-white selection) coupled to the limonene generation system consisting of dxs (deoxyxylulose phosphate synthase, rate controlling step of terpenoid biosynthesis in bacteria) + LIMS1 (limonene synthase 1 from lemon) + appY (a transcription factor known to increase terpenoid biosynthesis).
The lac promoter, dxs and appY are from E. coli; LIMS1 is from lemon.
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.
crtI (phytoene dehydrogenase) and crtB (phytoene synthase)
phytoene dehydrogenase and phytoene synthase from Pantoea ananatis (formerly Erwinia uredovora) DSMZ 30080 (ATCC 19321). Accession: D90087. Part of the carotenoid biosynthesis pathway. Note: the crtI coding sequence contains 2 PstI sites. In a later version of this part, these have been removed by site-directed mutagenesis but the functionality of the nutated gene has not been confirmed. This version still contains the two PstI sites. 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)
Note: the crtI coding sequence contains 2 PstI sites. In a later version of this part, these have been removed by site-directed mutagenesis but the functionality of the nutated gene has not been confirmed. This version still contains the two PstI sites. Note also that the crtB coding sequence overlaps the crtI coding sequence; for simplicity, in this version, they have not been separated.
crtE from Erwinia uredovora with a rbs preceeding it. This gene codes for geranylgeranyl diphosphate synthase which is the first committed step in the beta carotene synthesis pathway.
We wanted to use this in synthetic operon construction, and as the first gene in the transcript, so we included a NdeI restriction site at the start codon.
The plasmid pAC-3.10.10.A which was sent to us by Ethan Johnson on iGEM Minnesota team timebomb.
This is the coding sequence of crtB from Pantoea ananatis (formerly Erwinia uredovora) (Accession number D90087) preceded by an E. coli synthetic ribosome binding site (rbs). It encodes phytoene synthase, part of the carotenoid biosynthesis pathway, which converts geranylgeranyl diphosphate to phytoene.
rbs: Escherichia coli JM109 genomic DNA. crtB: Pantoea ananatis genomic DNA
This is the coding sequence of ''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.
The synthetic ribosome binding site was added by fusion PCR to the ''crtY'' gene, which was amplified from ''P. ananatis'' genomic DNA.
This is the first enzyme in the mevalonate pathway we want to introduce into E. coli. It is needed to turn acetyl-coA into acetoacetyl-coA. The enzyme itself originates from E. coli and is called AtoB (AE014075). An alternative to this would be ERG10 from S. Cerevisiae.
B-carotene monooxygenase, Produces retinal from B-carotene. ninaB gene from Drosophila.
This coding region encodes a beta-carotene deoxygenase, that splits beta-carotene into two retinal molecules.
No considerations have been made so far concerning design
dxs coding sequence encoding 1-deoxyxylulose-5-phosphate synthase
This is the coding sequence of dxs from Escherichia coli JM109. It encodes 1-deoxyxylulose-5-phosphate synthase, thiamine-requiring, which catalyses teh first step in the biosynthesis of terpenoids. Overexpression of dxs has been reported to increase yields of carotenoids and other terpenoids [add reference here].
3-Hydroxy-3-Methyl-Glutaryl-CoA-Reductase from Yeast
This is a truncated version of the 3-Hydroxy-3-Methyl-Glutaryl-Coenzyme-A-Reductase (HMG-CoA-R) from ''Saccharomyces cerevisiae''. It catalyses the reaction of 3-Hydroxy-3-Methyl-Glutaryle-Coenzyme-A to Mevalonate which occurs in the Mevalonate Pathway to Isopentylpyrophosphate which is the Precurser of all Isoprenoides. The enzyme is naturally a membrane bound enzyme. The membrane anchor is situated N-terminal. This region is also essential for the degradational control of the enzyme. By deleting the N-terminal part to Aminoacid (inclusive) the enzyme is expected to be water soluble and deregulated.
The truncation was done according to the following publication: Rico, Pardo, Orejas: 'Enhanced Production of a Plant Monoterpene by Overexpression of the 3-Hydroxy-3-Methylglutaryl Conenzyme A Reductase Catalytic Domain in Saccharomyces cerevisiae'; Applied and Enviromental Microbiology; Vol 76 p. 6449-6454; 2010
The original source of this part is the genome of Saccharomyces cerevisiae. We obtained the sequence by de novo synthesis. For that we used data from the Saccharomyes Genome Database. We inserted some calm mutations to avoid conflicts with the Cloning Standard 10.
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.
Expresses B-carotene monooxygenase on a constitutive promotor
This generator expresses a beta-carotene 15,15'-monooxygenase,on a constitutive promotor, that catalyses retinal production from beta-carotene via the following reaction: beta-carotene + O2 = 2 retinal
The brick requires B-carotene supply to function. This can either be supplied externally, or alternatively the BBa_K274210 B-carotene generator will induce full B-carotene synthesis in E. coli.
The ninaB gene was originally cloned from drosophila melanogaster, and cDNA was aquired for production of the biobrick from the Drosophila Genome Ressource Center. Promotor+rbs brick BBa_J13002 and terminator BBa_B0015 were used.
crtE from Erwinia uredovora with a rbs preceeding it. This gene codes for lycopene cyclase which is the third step in the beta carotene synthesis pathway. In combination with CrtE and B, this works to produce lycopene, a red, antioxidant, carotenoid.
We needed to use site directed mutagenesis to add the SpeI site after the stop codon.
The plasmid pUC-EUY which was sent to us by Ethan Johnson on iGEM Minnesota team timebomb.
bifunctional Cyclase from Gibberella fujikuroi
Genomic sequence of Gibberella fujikuroi.
Promoterless Synthetic Operon of CrtE,B,I,Y and GFP
This operon provides the full set of bacterial open reading frames necessary for beta carotene production with a GFP and termination stop signals tacked on at the end for good measure! crtE,B,I,Y coding regions from Erwinia uredovora each with a rbs preceeding the open reading frame. Likewise, the RBS-GFP-Term from biobrick BBa_E0240 is included on the end to provide a visual signal affirming transcription of preceeding 4 genes. There is no promoter on this construct, and after this GFP biobrick, no more genes can be added.
The synthetic operon needed a transcriptional termination signal, as well as GFP to visualize complete transcript production, so BBa_E0240 was used. No more genes can be added to this operon with this GFP on the end.
This operon is made from biobricks BBa_K152001 - BBa_K152003, the biobrick BBa_K118005 which was sent to us by iGEM Edinburg, and BBa_E0240.
This is the coding sequence of crtI from Pantoea ananatis (formerly Erwinia uredovora) (Accession number D90087) preceded by an E. coli synthetic ribosome binding site (rbs). It encodes phytoene dehydrogenase, part of the carotenoid biosynthesis pathway, which converts Phytoene to Lycopene.
rbs: Escherichia coli JM109 genomic DNA. crtI: Pantoea ananatis genomic DNA
ent-Kaurene Oxidase from Gibberella fujikuroi
Genomic Sequence from Gibberella fujikuroi