1887

Abstract

Temperature sensitivity is often used as a way to attenuate micro-organisms to convert them into live vaccines. In this work, we explore the use of temperature-sensitive (TS) genetic circuits that express lethal genes as a widely applicable approach to TS attenuation. We tested different combinations of TS repressors and cognate promoters controlling the expression of genes encoding restriction endonucleases inserted at four different non-essential sites in the chromosome. We found that the presence of the restriction endonuclease genes did not affect the viability of the host strains at the permissive temperature, but that expression of the genes at elevated temperatures killed the strains to varying extents. The chromosomal insertion site of the lethal cassettes affected their functionality, and insertion at one site, , rendered them ineffective at inducing death at high temperature. Induction of a TS circuit in a growing culture led to a reduced cell mass and a reduction of the number of cells that could exclude a dye that indicated viability. Incubation of cells carrying a TS lethal gene circuit initially grown at low temperature and then suspended in phosphate buffered saline at high temperature led to about 100-fold loss of cell viability per day, compared to a minimal loss of viability for the parental strain. Strains carrying either one or two TS lethal circuits could generate mutants that survived at high temperature. These mutants included complete deletions of the lethal gene circuits.

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2017-04-01
2019-12-08
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