Team:Johns Hopkins-Wetware/Parts

We constructed and submitted three vectors that can easily convert our Yeast Golden Gate Parts into BioBricks. These conversion vectors consist of the pSB1C3 standard BioBrick backbone and also contain RFP (BBa_J04450) for easy red/white screening. We have inserted BsaI sites flanking the RFP gene. BsaI digestion exposes the signature overhangs corresponding to a promoter, coding sequence or terminator(BBa_K799024,BBa_K799025,BBa_K799026) In a one-pot digestion-ligation reaction, Yeast Golden Gate Parts can be converted to BioBricks through a red/white selection.

We have sequenced these vectors across the RFP insert to verify the signature overhang and BsaI sequences. Using these new vectors, have also performed one-pot digestion ligation reactions to successfully convert 5 promoters, 2 ORFs, and 1 terminator sequence into BioBrick-compatible parts.

Using the conversion vectors described above, we BioBricked five ethanol-inducible yeast promoters (BBa_K799001, BBa_K799003, BBa_K799005, BBa_K799007, BBa_K799009), human cytochrome p450 2E1 (CYP2E1:BBa_K799027), and the yeast terminator from the MFA2 gene (BBa_K799029). We also BioBricked a GFP sequence lacking BsaI and BsmBI(BBa_K799028)that we constructed for use in our Yeast Golden Gate Assembly method. We have sequence verified all of these parts. The ethanol-inducible promoters have been characterized through GFP expression under various ethanol levels. We have tested the ability of CYP2E1 to reduce ethanol concentration over a 24 hour fermentation.

In the first iteration of The Yeast Golden Gate Parts Course, we generated 831 parts including promoters, ORFs, and terminators. Importantly, these parts can be converted to the BioBrick standard using our conversion vectors described above (spreadsheet of all made parts). All of these parts derive from the Saccharomyces cerevisiae genome and information about them can be found at Saccharomyces Genome Database.