Tools for Synthetic Biology

Project Summary

We have developed a variety of synthetic biology tools to facilitate metabolic engineering in both yeast and E. coli. Examples include our expression and integration vectors. The pCEV vectors allow over-expression of multiple genes in yeast using antibiotic resistance for selection. This is particularly useful for industrial strains that do not have engineered auxotrophies, or for heavily engineered strains that have no auxotrophic markers remaining for selection. We also have a vector series for rapid, efficient integration of very large DNA sequences onto the E. coli genome. These knock-in/knock-out (KIKO) vectors are particularly useful for introduction of multiple genes, for example when reconstructing long metabolic pathways. We also developed a method to reliably engineer the sucrose utilization phenotype in (previously) non-sucrose-utilizing E. coli strains. The pCSCX plasmid can be used to rapidly transfer a permease-mediated system onto most E. coli genomes.

Regulating gene expression at appropriate times during cultivation is very important to help avoid/mitigate problems of metabolic burden (excessive competition between production of the biochemical of interest and core metabolism required for cell growth) and/or product toxicity. We have developed synthetic biology circuits to help control appropriate expression patterns. The native yeast system, naturally used to detect cell density (‘quorum sensing’) was hi-jacked to interface with signal amplification and regulation systems, so that sharp switch-like control of gene expression is achieve in response to increased cell density. This engineered quorum-sensing system is now being applied to a variety of problems.

Plasmids are available from AddGene.