Mycobacterium tuberculosis remains one of the most common worldwide causes of illness and death due to an infectious disease. The emergence of multiple and extreme-drug resistant strains has increased the need to find an effective vaccine for tuberculosis. The goal of our research group is to engineer a temperature-sensitive (TS) M. tuberculosis strain that can be used as a tool in vaccine development. One approach to create TS M. tuberculosis involves the integration of the essential gene ligA encoding a TS NAD+ dependent DNA ligase, which was taken from the psychrophilic organism Pseudoalteromonas haloplanktis. The integration and functioning of ligA was demonstrated in the fast-growing organism Mycobacterium smegmatis. This strain had a TS phenotype with growth limited to below 37°C. The strain was found to have a stable TS phenotype and did not mutate to a temperature-resistant form at a detectable level. Following experiments with the fast growing M. smegmatis, the integration of the ligA gene was attempted in slow-growing M. tuberculosis. Merodiploids of M. tuberculosis containing both the psychrophilic and the WT ligA gene in its chromosome were obtained.
The second approach used for the development of TS M. tuberculosis was the directed evolution of native M. tuberculosis essential genes. An advantage of this approach is that the gene encoding the essential protein will resemble the native M. tuberculosis gene and thus will closely match the native transcriptional and translational rates. A system to screen and select for TS essential genes engineered by directed evolution was designed, where the essential gene on the chromosome of E. coli was knocked out and this gene was supplied on a conditionally replicating plasmid. As a first step in developing this directed evolution approach, a family of conditionally replicating plasmids were created and tested in an essential gene knock-out strain of E. coli. / Graduate
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/6280 |
| Date | 29 June 2015 |
| Creators | Pinto, Crystal Tina |
| Contributors | Nano, Francis E. |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Rights | Available to the World Wide Web |
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