A Temperature-controlled Gene Expression System in
Escherichia coli
Abstract:
Conditional expression of desired gene can be of great importance when the gene product is needed only at the specific circumstance. In this seminar, our recent studies on the non-canonical amino acid (ncAA) auxotrophic bacterial strain will be discussed. Specifically, the main contents will include the engineering of an orthogonal protein-protein interface and selection protocol that can be generally used to construct an ncAA dependent bacterial strain. Additionally, an ncAA- and temperature-controlled gene expression system will be introduced. We recently have identified conditions to regulate the expression of the genes of interest (GOI) in an Escherichia coli cya that lacks cyclic AMP production capability. Expression of exogenous tRNA in E. coli cya disables cAMP dependent protein expression at 37 °C, whereas the ability for protein expression is restored at lower temperatures below 30 °C. To provide more stringent conditions for GOI expression, fusions with an ncAA p-benzoylphenylalanine (BzF) dependent orthogonal protein-protein interface and split units of Bordetella pertussis adenylate cyclase were used. The system exhibited the BzF dependence and temperature sensitivity for expression of GOIs including chloramphenicol acetyltransferase and super-folder green fluorescent protein. All the gene expressions were induced well at below 30 °C and 0.5 mM BzF conditions, while suppressed at 37 °C or without BzF condition.
References
[1] M. Koh; F. Nasertorabi; G. W. Han; R. C. Stevens; P. G. Schultz, J. Am. Chem. Soc., 2017, 139 (16), 5728-5731.
[2] M. Koh; A. Yao; P. R. Gleason; J. H. Mills; P. G. Schultz, J. Am. Chem. Soc., 2019, 141 (41), 16213-16216.
[3] G. Karimova; J. Pidoux; A. Ullmann; D. Ladant, Proc. Natl. Acad. Sci. USA, 1998, 95 (10), 5752-5756.