Synthetic operon of CrtEBIY and FrdBCD and GFP
A limiting factor behind carotenoid accumulation in many organisms is sink strength. Carotenoids are lipophilic, so it is hoped that increased membrane might provide more storage capacity for beta carotene accumulation. In addition to the four Crt genes required for beta carotene production, three of the four genes in the FrdABCD operon were included to increase membrane production, while GFP was added to visualize transcript production and ensure transcription termination.
This operon contains 8 genes and was harder to put together than smaller constructs. Nothing specific though.
rbs: Escherichia coli JM109 genomic DNA. crtI: Pantoea ananatis genomic DNA
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.
The plasmid pAC-3.10.10.A which was sent to us by Ethan Johnson on iGEM Minnesota team timebomb.
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.
We got this from the plasmid pAC-3.10.10.A sent to us by Ethan Johnson of iGEM Minnesota team timebomb.
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.
The plasmid pUC-EUY which was sent to us by Ethan Johnson on iGEM Minnesota team timebomb.
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.
frdBCD operon from E. coli (missing the first gene fumarate oxidase)
This partial operon contains the three operon reading frames found after the large fumarate oxidase genes. Fumarate reductase catalyzes the reduction of fumarate to succinate in the terminal step of anaerobic electron transfer to fumarate and is a membrane-bound complex composed of four different polypeptides designated FrdA, FrdB, FrdC, and FrdD. FrdA is a large protein that protein binds flavin adenine dinucleotide. FrdB contains the reactive iron-sulfur cofactors, while FrdC and FrdD are membrane proteins which bind FrdA and B to the inner side of the cytoplasmic membrane. J Bacteriol. 1987 July; 169(7): 3340???3349. Overexpression of FrdABCD results in a doubling of E. coli cytoplasmid membrane (J Bacteriol. 1984 May; 158(2): 590-596), so it is hoped overexpression of FrdBCD will also increase membrane area.
FrdA was not included in the production of this biobrick, on account of it containing several Biobrick sites; too many to remove through site directed mutagenesis. Hopefully FrdBCD is enough to induce the membrane doubling phenotype.
Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 <genbank>V01146</genbank>.
Transcription terminator for the E.coli RNA polymerase.
Suggested by Sri Kosuri and Drew Endy as a high efficiency terminator. The 5' end cutoff was placed immediately after the TAA stop codon and the 3' end cutoff was placed just prior to the RBS of Gene 1.4 (before AAGGAG). Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator.
RBS.3 (medium) -- derivative of BBa_0030
Weak1 RBS based on Ron Weiss thesis. Strength is considered relative to <bb_part>BBa_B0030</bb_part>, <bb_part>BBa_B0031</bb_part>, <bb_part>BBa_B0033</bb_part>.
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix ("RBS-2" in figure 4-14 of thesis). Contact info for this part: Brian Chow
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
Transcriptional terminator consisting of a 64 bp stem-loop.
green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (SwissProt: P42212
GFP (mut3b) [note that this part does not have a barcode]
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.
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.
Conversion of the iGEM parts registry to SBOL2.1