A translation-coupling DNA cassette for monitoring protein translation in Escherichia coli

Metabolic Engineering
Daniel Mendez-PerezBrian F Pfleger

Abstract

A major challenge to using heterologous expression in metabolic engineering experiments is the inability to quickly dissect experiments that have failed at the stage of translating mRNA. While many methods of detecting proteins exist, methods that detect untagged proteins at low levels are limited. Here, we describe a method to quickly determine whether Escherichia coli is capable of expressing the product of any target gene by coupling translation of a target gene to a detectable response gene. A translational coupling cassette was designed to encode a mRNA sequence that forms a secondary structure in the absence of translation and contains the translational start sequence of a detectable response gene. The translational coupling method was successfully tested with fluorescent proteins and antibiotic resistance markers. Only when the target gene was fully translated was the response observed. Further characterization demonstrated that translational coupling functions at both low and high levels of expression and that the response signal is proportional to the amount of target gene product. The translational coupling system was used to determine that a large multi-domain enzyme was not actively translated in E. coli, to isolate...Continue Reading

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Citations

Nov 8, 2014·Computational and Structural Biotechnology Journal·Amanda K FisherRyan S Senger
Apr 2, 2013·Current Opinion in Biotechnology·Rebecca M Lennen, Brian F Pfleger
Mar 10, 2018·Microbial Cell Factories·Roberto FerroMorten H H Nørholm
Dec 31, 2019·Molecular Systems Biology·Belén CallesVíctor de Lorenzo

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