Abstract : Rapid, Flexible, and Affordable Yeast Genome Engineering with BioBrickTM Standardization

The BioBrickTM Standard 10 was updated to allow site-directed chromosomal modification in E. coli. This design is incompatible with Saccharomyces cerevisiae. In this project, we develop a library of flexible plasmids that adhere to the Standard and Type IIS Assembly and that allow for the systematic and efficient cloning of a desired gene within target yeast chromosomal loci, while maintaining the plasmid’s compatibility with E. coli. Our plasmid library targets the Ade2, His3, Ade4, and Gal4 loci, and is equipped with KanMX, NatMX, Ura3, and His3 yeast-selectable markers as well as RFP to enable colorimetric selection in E. coli. We use our experimental procedure to develop simple protocols to allow individuals with minimal laboratory experience to reproduce and expand our library. Finally, we endeavour to expedite and reduce the cost of cloning by making a DIY Gibson Assembly kit, in which all the required enzymes are harvested in the laboratory.