In part, due to a lack of terminator characterization for applications in the fields of metabolic engineering and synthetic biology, most laboratories use a limited number of terminators for heterologous protein expression. This poses a problem when engineering longer metabolic pathways, as repeated terminator sequences can promote undesired recombination events. A larger selection of characterized terminators also provides an opportunity to further regulate gene expression. We cloned 16 terminators from the genome of Saccharomyces cerevisiae (baker's yeast) and characterized their impact on expression with a fluorescent reporter driven by a variety of promoters, including the strong TDH3 promoter. Fluorescence varied by 190 fold between terminators regulated by the TDH3p with NAT1 terminator yielding the most protein expression.  Furthermore, promoters and terminators appear to mostly impact protein expression independently, based on a screen of six terminators driven by TDH3p and three other constitutive promoters: TEF1p, RPL18Bp, and REV1p.

Optomizing gene expression by characterizing terminators in Saccharomyces cerevisiae

247th National Meeting (2014)

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