Temperature-sensitivity (TS) is often used as a way to attenuate microorganisms to convert them into live vaccines. Studies indicate that live vaccines are often necessary for the complete clearance of certain pathogenic organisms. In this work we explore the use of TS genetic circuits that express lethal genes for their potential utility as a widely applicable approach to TS attenuation. Here, we use restriction endonucleases as the lethal gene products. 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 Escherichia coli 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 location of the genetic circuit cassette in the chromosome was critical, and insertion at the ycgH site led to minimal cell death. Induction of the TS circuit in a growing culture led to a pre-mature leveling off of the optical density, and a shift in the number of cells that could exclude a dye that indicated cell viability. Incubation of cells 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 minimal loss of viability for the parental strain. The Dual strain containing two different genetic circuits was found to have reduced escape frequency compared to single circuit strains. However, strains carrying either one or two TS lethal circuits could generate mutants that survived high temperature. These mutants included start codon deletions as well as upstream deletions of the TetRD1 encoding gene as well as complete deletions of the lethal gene circuits. / Graduate
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/7503 |
| Date | 30 August 2016 |
| Creators | Pearce, Stephanie |
| Contributors | Nano, Francis E. |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | Available to the World Wide Web, http://creativecommons.org/licenses/by-nc-nd/2.5/ca/ |
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