Promoterless Synthetic Operon of CrtE,B,I,Y and GFP
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 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.
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.
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.
Conversion of the iGEM parts registry to SBOL2.1