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Violacein
We plan to finish ligating the 5 violacein expression cassettes into the PRS416 Yeast Vector and transform it into a URA3 Knockout yeast strain in nutrient-rich media lacking Uracil. Our library of Golden Gate assembly compatible yeast promoters and terminators will then include the entire violacein pathway (via BsmBI digestion). In the future, this pathway can be used to test novel neochromosomes & complex DNA structures in yeast by selecting for purple colonies.
We plan to finish the violacein loxPsym integration project. We will transform the RPS2 3'UTR into E. coli, thus completing our library of Golden Gate Assembly-compatible yeast promoters and 3'UTRs. We will then combine our promoter/3'UTR library with the violacein genes and a loxPsym linker in a single ligation reaction, where the promoters, 3'UTRs, and violacein ORFs will randomly combine into expression circles with loxPsym sites. The expression circles will then be transformed into the yeast strain containing synIXR, which will be grown up in the presence of estradiol. Estradiol will induce expression of the Cre-EBD fusion protein, which will catalyze loxP recombination, allowing some yeast cells to take up all five violacein genes and begin synthesizing violacein in various amounts depending on the promoters and 3'UTRs that were attached to the violacein ORFs in the random ligation.
Characterizing the Promoters
We have successfully transformed a number of promoters (BAP2p, TDH3P, KRE9p, HHO1p, PRY1p). We have also successfully transformed reference promoter constructs into yeast. We plan to proceed to flow cytometry analysis in the near future. We also plan to transform our other promoters into yeast. We hope to conclude this part of the project before the Americas Regional.
