title: A translation-coupling DNA cassette for monitoring protein translation in Escherichia coli.
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 the translation problems to the C-terminal domains, and to optimize conditions for expressing a codon-optimized sequence variant.
authors.affiliation: University of Wisconsin-Madison, Department of Chemical and Biological Engineering, 1415 Engineering Dr., Madison, WI 53706, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Mendez-Perez
authors.forename: Suman
authors.initials: S
authors.lastname: Gunasekaran
authors.forename: Victor J
authors.initials: VJ
authors.lastname: Orler
authors.forename: Brian F
authors.initials: BF
authors.lastname: Pfleger
mesh_terms: D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression; D060847:Metabolic Engineering / Q000379:methods*; D009841:Oligonucleotides / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D014176:Protein Biosynthesis
chemical_list: D029968:Escherichia coli Proteins; D009841:Oligonucleotides; D004247:DNA