Catalytically active CYP6G1 coding device
CYP6G1 comes from Drosophila melanogaster genome and fCPR comes from Musca domestica.
This device was constructed to produce catalytically active CYP6G1 in B. subtilis and secrete it outside. Secretion is needed since CYP6G1 enzyme should degrade imidacloprid in bee midgut. To provide constitutively production and secretion of this enzyme, we used Pveg constitutive promoter, SpoVG RBS with SacB signal peptide (K541501) which is used by Fatih_Turkey Team in 2011. Also fCPR production is necessary to make CYP6G1 enzyme catalytically active. Thus, SpoVG RBS and SacB signal peptide was used before fCPR too.
Promoter and RBS are specific for expression and signal peptide is specific for secretion in B. subtilis.
Double terminator consisting of BBa_B0010 and BBa_B0012
It is obtained from Drosophila Melanogaster genome and modified according to work in B.subtilis.
It is an insecticide resistance gene which is found in Drosophila Melanogaster. We used it in our system to metabolize imidacloprid by producing nontoxic byproducts. It is obtained from Drosophila Melanogaster genome and modified according to work in B.subtilis. Original sequence was modified since it was not proper for prokaryotic expression. Its first 21 nucleotide which coding 7 aa was changed with "Barnes" sequence: MVLTEVL is the native aa sequence, and MALLLAV is the Barnes aa sequence. (Barnes et al. 1991) Reference: Barnes, H.J., Arlotto, M.P., Waterman, M.R., 1991. Expression and enzymatic activity of recombinant cytochrome P450 17a-hydroxylase in Escherichia coli. Proc. Natl. Acad. Sci. USA 88, 5597e5601.
Original sequence was modified since it was not proper for prokaryotic expression. Its first 21 nucleotide which coding 7 aa was changed with "Barnes" sequence: MVLTEVL is the native aa sequence, and MALLLAV is the Barnes aa sequence. (Barnes et al. 1991) Reference: Barnes, H.J., Arlotto, M.P., Waterman, M.R., 1991. Expression and enzymatic activity of recombinant cytochrome P450 17a-hydroxylase in Escherichia coli. Proc. Natl. Acad. Sci. USA 88, 5597e5601.
The source of fCPR is Musca domestica genome, and SacB comes from B. subtilis.
fCPR is a cytochrome P450 reductase which makes CYP6G1 enzyme catalytically active. And SacB is a signal peptide in B. subtilis which directs produced proteins outside of the cell.
SacB sequence was added to upstream of fCPR sequence to create a fusion protein which secrete outside of the cell.
Promoter Pveg, RBS spoVG and SacB signal peptide for B. subtilis
All three basic components come from B.subtilis genome
Constitutive promoter veg(BBa_K143012) coupled to the strong Ribosome Binding Site spoVG(BBa_K143021) from B. subtilis. These parts have been taken from 2008_Imperial_Collage. Pveg is a constitutive promoter that constitutively expresses the P43 protein in B. subtilis. SpoVG is an endogenous ribosome binding site from B. subtilis. The sequence of the spoVG ribosome binding site is AAAGGUGGUGA which is complementary to the sequence UUUCCUCCACU from the 3' region of the 16s rRNA from B. subtilis. SacB is a signal peptide used in the Sec-SRP (secretory signal recognition particle) pathway by B. subtilis. Signal peptides are responsible for directing preproteins (secretory proteins with a signal peptide region attached) through an appropriate secretory pathway. In the case of the Sec-SRP signal peptide, they direct preproteins from the cytoplasm into the growth medium.
SpoVG Ribosome Binding Site (RBS) for B. subtilis
SYnthesized RBS because part BBa_K143021 was wrong.
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