Team:RHIT/Project/Wetlab

• 
• 
  HOME
• 
  TEAM
  • Team Members
  • Attributions
  • Official Team Profile
• 
  PROJECT
  • Wetlab
  • Human Practices
  • Sustainability
• 
  NOTEBOOK
  • Protocols
  • Journal
  • Parts
  • References
• 
  SAFETY
• 

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 promoting

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

The second open reading frame of this yeast construct includes constitutive promotion of lactate dehydrogenase (LDH). LDH allows for the yeast to produce lactate. The lactate produced by the yeast will induce E. coli's lactate promoter, which serves to activate a His3 gene that will enable the E. coli to survive without supplemental histidine in the media (See above).

To test our transformed E. coli strain for the surface expression of alpha-mating factor and the ability to induce the MPRP, Team RHIT designed a positive feedback mechanism that expresses blue fluorescent protein within yeast. We again use the FUS1 promoter because it is activated by the mating pheromone response pathway. Upon FUS1 promoter activation, the blue fluorescent protein (BFP) expression begins. To further positive feedback, the LexA binding domain binds to the LexA regulatory element and, in conjunction with the VP64 (viral protein 64) transcriptional activator domain, facilitates further production. The presence of two BFP domains, along with the glysine-serine linkers (GSLs) that link the two domains, ensure a good output of fluorescent protein. In addition, a nuclear localization signal (NLS), whish is a short amino acid sequence, was included to "tag" the protein for import into the nucleus by nuclear transport. A bright blue dot in the yeast nucleus will show a positive test result.

Hypothesis

Team RHIT hypothesizes that the synthetic unity system will induce a symbiotic relationship between yeast and E. coli. This symbiosis will develop a novel design chassis that will spur innovation within synthetic biology.