Team:Johns Hopkins-Wetware/Parts

We constructed and submitted 3 vectors to easily convert our Yeast Golden Gate Parts into BioBricks, one each for converting promoters, coding sequences, and terminators (BBa_K799024, BBa_K799025, BBa_K799024). These conversion vectors consist of the backbone standard BioBrick registry shipping vector pSB1C3 containing the standard BioBrick vector backbone part, which codes for RFP (BBa_J04450). The RFP is flanked by signature overhangs corresponding to a promoter, coding sequence, or terminator. 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 and signature overhangs to verify these parts, and have used them to successfully generate the BioBricks below.

We BioBricked 5 ethanol-induced yeast promoters with varying induction levels (BBa_K799001, BBa_K799003, BBa_K799005, BBa_K799007, BBa_K799009), the human cytochrome p450 2E1 (CYP2E1) used in our ethanol-control project (BBa_K799027), and the yeast terminator from the MFA2 gene (BBa_K799029). We also BioBricked a GFP sequence lacking BsaI and BsmBI sites for use with Yeast Golden Gate (BBa_K799028). 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 our BioParts course, we generated around 850 yeast parts including promoters, coding sequences, and terminators. These are parts that can be converted to the BioBrick standard using our conversion vectors above.