crtZ (beta-carotene hydroxylase) with native rbs
crtZ (beta-carotene hydroxylase) from Pantoea ananatis (formerly Erwinia uredovora) DSMZ 30080 (ATCC 19321). Accession: D90087. Part of the carotenoid biosynthesis pathway. Converts beta-carotene to the yellow pigment zeaxnathin.
This part was made as a single biobrick rather than separate rbs and coding sequence, hence the use of blunt combination to generate the final sequence.
Pantoea ananatis (formerly Erwinia uredovora) DSMZ 30080 (ATCC 19321). Accession: D90087.
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.
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.
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.
rbs: Escherichia coli JM109 genomic DNA. crtI: 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.
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].
COMT (caffeic acid-O-methyl transferase) with ribosome binding site. Enzyme methylates -OH on C4 of the caffeic acid aromatic ring to produce ferulic acid. COMT is the third gene in the vanillin biosynthesis pathway,
Used COMT cDNA as genomic DNA contains introns
Genes and Enzymes Involved in Caffeic Acid Biosynthesis in the Actinomycete Saccharothrix esanaensis, Berner M, et al, Journal of Bacteriology 188(7): 2666, (2006) Gowri G, et al, Molecular cloning and expression of alfalfa S-adenosyl-L-methionine: caffeic acid 3-0-methyltransferase, a key enzyme of lignin biosynthesis, Plant Physiology 97(1):7 (1991) Downregulation of Caffeic Acid 3-O-Methyltransferase and Caffeoyl CoA 3-O-Methyltransferase in Transgenic Alfalfa Impacts on Lignin Structure and Implications for the Biosynthesis of G and S Lignin, Guo D, et al, Plant Cell. January; 13(1): 73 (2001)
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.
rbs+glgC (ADP-glucose pyrophosphorylase)
This is the coding sequence of ''glgC'' from ''Escherichia coli'' JM109 with added ribosome binding site (rbs). It encodes ADP-glucose pyrophosphorylase, which catalyses Glucose 1-phosphate + ATP -> ADP-glucose + PPi, a step in the production of glycogen from glucose.
This part is adapted from K283006 with a terminator added to the end of the device.
The CI, terminator, pLambda promoter and the chez coding region are self designed The rbs, and the pLux promoter are from the registry
ftsK (cell division protein) of E. coli with native rbs
This is cell division gene ftsK of Escherichia coli JM109 (a K12 strain) with its native ribosome binding site. Note that this is the native sequence with a TTG start codon and two internal PstI sites.
Note that this is the native sequence with a TTG start codon and two internal PstI sites.
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.
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
rbs+glgC16 (glgC with G336D substitution)
This is the coding sequence of ''glgC'' (ADP-glucose pyrophosphorylase) from ''Escherichia coli'' JM109 with the substitution G336D, with added ribosome binding site (rbs). This mutation is known to cause increased activity of ADP-glucose pyrophosphorylase in the absence of the activator fructose 1,6-bisphosphate (FBP), high affinity for FBP and substrates lower affinity for the inhibitor AMP. (Leung ''et al.'', 1986; Meyer ''et al.'', 1988)
Limonene synthase gene with ribosome binding site
Ribosome binding site attached to Limonene synthase gene. Limonene synthase enzyme produces (+)-limonene from farnesyl diphosphate (intermediate in mevlonate pathway). (+) Limonene is a component of lemon scent.
several codons were altered silently to remove several forbidden restriction sites. The N-terminal region (plastid targeting sequence) was removed, removing the first 51 amino acids and replacing codon 52 with the start codon ATG. LIMS cDNA was used as genomic DNA contains introns.
Monoterpene biosynthesis in lemon (Citrus limon) cDNA isolation and functional analysis of four monoterpene synthases, J Lucker, et al, Eur. J. Biochem. 269: 3160???3171 (2002) Analysis of Lemon and Bergamot Essential Oils by HPLC with Microbore Columns, M Benincasa, et al, Chromatographia 30(5/6):271 (1990) Citrus fruit flavour and aroma biosynthesis, L Sharon-Asa, The Plant Journal, 36:664 (2003)
sam5 (coumarate hydroxylase) with native rbs.
Sam5 (4-coumarate-3-hydroxylase) of the bacterium Saccharothrix espanaensis DSM 44229 with its native rbs.
This was made as a single biobrick, rather than separate rbs and coding sequence, hence the use of blunt ligation in generating this composite part.
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.
rbs+appY (encoding a DNA-binding transcriptional activator)
This is the coding sequence of appY from Escherichia coli JM109 preceded by a synthetic ribosome binding site (rbs). It encodes a transcriptional regulator related to anaerobic energy metabolism. Overexpression of the gene has been reported to result in enhanced lycopene production by E. coli (Kang et al., 2005).
sam8 (tyrosine ammonia lyase) with native rbs
This is sam8 (tyrosine ammonia lyase) of the bacterium Saccharothrix espanaensis DSM 44229 with its native ribosome binding site.