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

  • Daniel E. Agnew
  • Brian F. PflegerEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 765)


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 



Work in the authors’ lab was sponsored by the University of Wisconsin-Madison Graduate School. Daniel Agnew is the recipient­ of an NIH Biotechnology Training Program Graduate Fellowship (NIH 5 T32 GM08349).


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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Chemical and Biological EngineeringUniversity of Wisconsin-MadisonMadisonUSA

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