Project Overview

Introduction

2010 was a great year for the uOttawa team, we successfully streamlined protocols and methods for manipulating the budding yeast S. cerevisiae. We submitted a number of important BioBricks™ to the registry. Among the submissions were the two drug selection cassettes NatMX and KanMX6, a novel cloning-vector that allows for rapid integration of BioBricks™ into the Ade4 locus of S. cerevisiae, as well as a range of promoters and repressors that function in yeast. Building off of last year’s successes, the uOttawa Team focused primarily on three fundamental technologies.

Objective 1: Quantitative Characterization

We feel that in order for synthetic biology to truly adopt an engineering ethos and set itself apart from traditional molecular biology, the quantitative characterization of genetic elements needs to be addressed. To this end we set out to design a reference strain of S. cerevisiae that would allow for the characterization of individual parts in whatever experimental conditions necessary. The initial inspiration for how such a characterization scheme would work came from Kelly et al. (2009). In this paper, a method for measuring the activity of BioBrick™ promoters is put forward. Promoter activity was measured against an internal reference promoter under defined experimental conditions. The method proposed controls for such factors as plasmid copy number and selection marker derived effects.

We found the Kelly paper to be instructive; however, because of differences in our model organism we adopted several modifications in our method. Whereas, Kelly et al. use E. coli transformed with low copy number plasmids, we integrate all of our constructs directly into the yeast genome. By integrating into the genome we are assured that there is only one copy of each construct, obviating the need to control for copy-number. Secondly, we wanted to simultaneously measure transcription factor (TF) expression and activity of its cognate promoter. Finally, the inherent difficulties associated with manipulating eukaryotic organisms forced us to commit to always integrate into the same genomic loc.

The above considerations, and our extensive experience with yeast led us to the following design for a reference strain. The reference strain will have a full length Act1 promoter driving the expression of yBFP (yeast-codon-optimized blue fluorescent protein) integrated into the Ade2 locus, likewise a second copy of the Act1 promoter driving yEGFP (yeast-codon-optimized green fluorescent protein) expression will be integrated into the Ade4 locus (fig. 1).

Figure 1: The reference strain of S. cerevisiae. The BFP expressing construct is integrated into the Ade2 locus using 200 bp regions of homology and uses KanMX6 selection. The GFP expressing construct is integrated into the Ade4 locus using 200 bp regions of homology and uses NatMX selection.

Several design considerations went into this reference strain. Firstly, both Ade2 and Ade4 are genes in the adenine synthesis pathway, and are considered neutral deletions. That is, if the adenine synthesis pathway is disrupted in yeast, the yeast can be grown in media supplemented with adenine. This media supplies the yeast with the necessary adenine for normal growth. Secondly, by targeting the adenine synthesis pathway, we are more easily able to select the correctly transformed yeast on the basis of color phenotype.

So, since our starting yeast strain (BY4742) has a fully intact adenine synthesis pathway, they appear creamy-white in color. The first step to generating the reference strain is the transformation of our wild-type (WT) BY4742 with Ade2-KanMX6-pACT1-BFP-Ade2. Once the yeast is transformed the DNA-repair mechanisms homologous recombinates our construct into the Ade2 locus, and in so doing removes the native ADE2 gene. Our yeast is then plated on YPD-agar plates supplemented with the antibiotic G418 and lacking adenine. The KanMX6 selection cassette confers resistance to G418 and ensures that correctly transformed yeasts are viable. In addition to drug selection, yeast with either an Ade2 deletion or mutation exhibit a color phenotype when grown with adenine, resulting in red colonies (fig.2).