Team:Baltimore BioCrew/Composite Part

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COMPOSITE PARTS

• BBa_K7301013






• BBa_K7301012






• BBa_K7301003

BBa_K3301013

This construct contains the BT2269 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.

BBa_K3301012

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.

BBa_K3301003

Our killswitch was made up of 5 different parts. Four of them were directly from HKUST, whose construct we based our design on. Those four are:

1. BBa_K733001
2. BBa_K733004
3. BBa_K733002
4. BBa_K733003

We also replaced the terminator that HKUST used with a well characterized promoter, part BBa_B1002. The initial 4 basic parts were rearranged from their original order, so the consituitive promoter (BBa_K73301) was producing the toxin (BBa_K733004), while the xylose inducible promoter (BBa_K733002) was producing the antitoxin (BBa_K733003).