Team:London Biohackspace/experiments/aking-auxotrophic-yeast
A Leucine knockout wildtype S. cerevisiae brewing strain will be created through the use of two BioBrick parts LEU2-Pre and LEU2-Suf that we have had synthesized. These parts are designed to knockout the LEU2 gene in S. cerevisiae through the process of homologous recombination. Any gene of interest inserted between these two parts will therefore replace the LEU2 gene within S. cerevisiae creating an auxotrophic strain that can be selected via supplementation of the growth media with leucine. For the purposes of this experiment we will be inserting a KanMX resistance gene that provides resistance to the antibiotic Geneticin (G418). Once these three parts have been assembled into a single plasmid we will PCR amplify the sequence to create a linear sequence that can be used to transform a wildtype brewing S. cerevisiae strain which will subsequently become auxotrophic for the amino acid leucine. These strains can then be transformed with the Miraculin and Lycopene S. cerevisiae expression vectors containing the LEU2 selection marker.Experiments
CREATING LEUCINE AUXOTROPHIC S. CEREVISIAE STRAINS
Introduction
Materials and methods
Results
Discussion
