Team:RHIT/Project/Wetlab

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Team RHIT aimed to create a symbiotic relationship between S. cerevisae and E. coli. We transform E. coli with a system that utilizes a fusion gene of ice nucleation protein and alpha mating factor to express alpha mating factor on the surface of E. coli. E. coli will now have the ability to induce the mating pheromone response pathway (MPRP) within yeast. Yeast utilizes FUS1 as the final signaling molecule of the MPRP. Therefore, we use a FUS1 promoter to express histidine within yeast. This means that E. coli must have an interaction with the yeast to allow for yeast's survival. For this to be a true symbiotic relationship, yeast must provide E. coli with a means of survival. E. coli is able to survive in this system when yeast expels lactate because our E. coli system requires lactate to promote histidine expression. To test our E. coli for alpha mating factor, we developed another system for yeast. Again we use the FUS1 promoter, but in this case we promote the expression of blue fluorescent protein within yeast. This system allows us to again visually confirm that E. coli and yeast are able to interact using alpha mating factor and the mating pheromone response pathway.

Using the Ice Nucleation Protein-based surface display presented by the Penn iGEM team in 2012, Team RHIT displays a fusion of the Ice Nucleation Protein and alpha-mating factor. Alpha Yeast Mating Factor induces the MPRP and FUS1 signaling in a haploid a-type yeast strains. The synthetic induction of the MPRP by E. coli is the first vital step toward synthetic unity.

To establish the symbiosis, the expression of an essential amino acid, histidine, promotes upon the activation of the lactate inducible promoter. The lactate derived within yeast.

Yeast benefits from unity with E. coli because the final signaling molecule of the MPRP, FUS1, induces the vital promotion of histidine.