Experiments

CREATING LEUCINE AUXOTROPHIC S. CEREVISIAE STRAINS

Introduction

We aim to create auxotropic S. cerevisiae strains using two approaches. The first approach will use homologous recombination to knockout a key amino acid biosynthesis gene such as LEU2 which encodes the protein sequence for the enzyme Beta-isopropylmalate dehydrogenase (IMDH). Subsequent transformation with a plasmid capable of expressing the LEU2 gene along with any gene of interest will restore leucine biosynthesis and allow for selection of transformants containing this plasmid. The second approach will use RNA interference to down regulate the LEU2 gene to a sufficient level to make the strain auxotrophic without altering the leucine biosynthesis pathway itself. A further transformation and homologous recombination event using a DNA sequence designed to replace the LEU2 RNA knockout will therefore restore the strains capacity for leucine biosynthesis as well as incorporate a gene of interest into the chromosomal DNA. This approach can be expanded by incorporating an additional RNA-based knockdown for another amino acid such as Uracil (URA3 - Orotidine-5'-phosphate (OMP) decarboxylase) therefore creating an additional homologous recombination target site for further chromosomal insertion and subsequent strain selection. Using this approach it will be possible to alternate between uracil and leucine auxotrophy and allow for multiple chromosomal insertion events via these synthetic homolgous recombination/RNA interference sequences.

Creating a Leucine auxotroph
A linear DNA sequence consisting of any gene of interest GOI flanked by LEU2-Pre and LEU2-Suf homolgousmrecombination overlaps will incorporate the GOI into the chromosome. Transformed strains will loose the ability to synthesize the amino acid Leucine so that addition of Leucine into the growth media can be used as a selection marker.

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.

Materials and methods

Results

Discussion