Part:BBa_K3301012
Codon optimized B. theta gene BT2268 with 6xHis tag and T7 promoter.
This construct contains the BT2268 gene that has been codon optimized for E. coli. The gene, originating from the symbiotic gut bacteria Bacteroides thetaiotaomicron, produces a protein of the Mucus-Associated Functional Factor (MAFF) molecular family. In the microbiome, this protein is only expressed when B. theta is embedded in the mucosal lining and coated with IgA. Our project sought to remove the gene from this restricted environment and enable its production in oxygen-tolerant E. coli, regulated by an inducible T7 promoter. This would enable production of the protein on an as-needed basis, to serve as a supplement to the microbiome after it is depleted by illness or antibiotics. A 6xHis tag was added for protein isolation and detection. It should be noted that this construct was too long to be synthesized by IDT, so it was synthesized in two pieces and later joined with BamHI restriction sites. However, the star activity of BamHI prevented the gene from ligating properly; this problem persisted over repeated trials in various conditions. BamHI is not recommended in this scenario.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1789
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1232
Illegal BsaI.rc site found at 1688
Illegal SapI site found at 1607
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