Results: Part Characterization using Flow Cytometry

After being integrated into the yeast genome, parts were characterized individually by flow cytometry.

TALEs

• Initial characterization: TALE-mCherry constructs are present and expression is inducible by β-estradiol. Cells were grown with and without β-estradiol and fluorescence was compared to background levels using a control strain. Our flow cytometry data shows ~4 fold increase in fluorescence with β-estradiol, indicating TALE transcription with operating mCherry tag.

• Relationship between beta-estradiol concentration and TALE-mCherry expression: Cells were grown in varied β-estradiol concentrations and fluorescence was compared to background levels using a control strain. The induction curves are shown for four strains of yeast that produced different TALEs (16B, 16C, 20B, and 20C) with β-ED concentrations of 0.1nM through 1000nM

ACT1 reporters

• Initial characterization: ACT1-yEVenus constructs with no operators showed distinct constitutive gene expression (green). However, presence of operator sites in the 5' UTR knocked down constitutive expression almost to background levels. In lieu of these results, we decided to construct TEF1-promoter reporter constructs. TEF1 is a stronger constitutive promoter that we hoped would counteract the effects of the operator sequence on constitutive expression levels.

TEF1 reporters

• Initial characterization: The TEF1-yEVenus constructs with operator sites in the 5' UTR that were submitted as BioBricks showed expression levels significantly higher than background levels, and comparatively higher than the ACT1-yEVenus constructs. This makes them suitable for further experiments measuring cooperativity of repression. The shift in fluorescence for BBa_K1204021, the 3x16C binding site reporter construct is shown below.