Strain Engineering

Volume 765 of the series Methods in Molecular Biology pp 99-111


Optimization of Synthetic Operons Using Libraries of Post-Transcriptional Regulatory Elements

  • Daniel E. AgnewAffiliated withDepartment of Chemical and Biological Engineering, University of Wisconsin-Madison
  • , Brian F. PflegerAffiliated withDepartment of Chemical and Biological Engineering, University of Wisconsin-Madison Email author 

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Constructing polycistronic operons is an advantageous strategy for coordinating the expression of ­multiple genes in a prokaryotic host. Unfortunately, a basic construct consisting of an inducible promoter and genes cloned in series does not generally lead to optimal results. Here, a combinatorial approach for tuning relative gene expression in operons is presented. The method constructs libraries of post-­transcriptional regulatory elements that can be cloned into the noncoding sequence between genes. Libraries can be screened to identify sequences that optimize expression of metabolic pathways, multisubunit proteins, or other situations where precise stoichiometric ratios of proteins are desired.

Key words

Synthetic biology Promoter Operon Ribosome binding site Intergenic sequence Megaprimer PCR Metabolic engineering mRNA stability Transcription termination