Strong promoter + Strong RBS + GSR + 10x Histidine tag + Double terminator
the cDNA of GSR was ordered from OriGene. The promoter and rbs was from iGEM distribution kit (BBa_K880005) and His-Term was from an intermediate part we constructed (BBa_K1898500).
BBa_K880005 is used to provide a strong promoter and strong rbs to maximize protein output. The ORF includes GSR, or glutathione reductase that catalyses the conversion of glutathione disulfide to glutathione, and 10x Histidine tag for protein purification. BBa_B0015 consists of a double terminator to end transcription.
We did five silent point mutations on gsr to remove the two Pst1 and three EcoR1 internal cutting sites. The sequence was then sent to Missiontech for mutagenesis. Primers were designed to remove the stop codon from GSR and were synthesized by Tri-I. Our assembly method BioBrick RFC[10] shifted the open reading frame and created a stop codon in between GSR and 10x histidine tag. The sequence was sent to Missiontech to add two base pairs in between the two genes.
Strong promoter, strong RBS combination for high expression levels of proteins
It is a combination of strong promoter (J23100) and RBS (B0034).
-J23100+B0034 -Strong promoter, strong RBS combination for high expression levels of proteins Consensus constitutive promoter and RBS sequence-produces strongest possible expression.
Josh Atkinson, Mike Ferguson, and Ben Parker
the cDNA of GSR is ordered from Origene.
GSR codes for gluatathione reductase, an enzyme that catalyzes the conversion of glutathione reductase to glutathione. Glutathione is an antioxidant exists in humans, plants, fungi, animal that prevents cellular damage by reactive oxygen species.
The purchased GSR cDNA has two internal PstI and three EcoRI cutting sites. After making silent mutations to the sequence, we sent the cDNA to MissionBiotech for mutagenesis to remove these cutting sites
RBS (Elowitz 1999) -- defines RBS efficiency
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix. No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info for this part: Brian Chow
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
Double terminator consisting of BBa_B0010 and BBa_B0012
10x-Histidine (10x-His) Tag with double stop codon (TAATAA)
Constructed through oligonucleotide annealing
10x-Histidine tag with double stop codon TAATAA to allow for better extraction of tagged products and protein termination in a single part.
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.
Transcriptional terminator consisting of a 64 bp stem-loop.
Conversion of the iGEM parts registry to SBOL2.1