<p>The Gram-positive, soil dwelling bacteria of the genus <em>Streptomyces</em> produce greater than 50% of the clinically relevant antibiotics in use today. Thanks to the falling price of DNA sequencing, <em>Streptomyces</em> genomes are revealing that they encode more secondary metabolites (potential antibiotics) than they produce under standard laboratory conditions. By heterologously overexpressing the known pleiotropic regulators of antibiotic expression from <em>Streptomyces coelicolor</em> in several other <em>Streptomyces</em> species it has been shown that the secondary metabolite profile of these species can be influenced. While present-day methods of introducing genes (conjugation) and screening for antibiotics work well on a small scale, the low throughput nature of these protocols stand as a barrier to testing this hypothesis on a larger scale. The focus of the research presented here was to develop high throughput (HTP) methods of engineering and screening <em>Streptomyces</em>. With these two technologies in place, an attempt was to made to introduce three plasmids (pSET152-<em>ermE*</em>p-null, pSET152-<em>ermE</em>*p-<em>atrA</em> and pSET152-<em>ermE</em>*p-<em>lsr2<sub>NTD</sub></em>) into 120 wild-isolate <em>Streptomyces</em> species from the Wright Actinomycete Collection. Exconjugants were successfully obtained for all three plasmids in 48 species of <em>Streptomyces</em> and were screened for increased antimicrobial activity using a HTP, <em>lux</em>-based bioassay. Numerous strains showed increased antimicrobial activity but WAC00206, WAC00230 and WAC00263 with pSET152-<em>ermE</em>*p-<em>lsr2<sub>NTD </sub></em>showed the most promising improvement in antimicrobial activity. These hits have been designated as high priority for future investigation. These results suggest that HTP conjugation and the <em>lux</em>-based bioassay are powerful methods for introducing plasmids into and screening engineered streptomycetes.</p> / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/11095 |
| Date | 10 1900 |
| Creators | Gverzdys, Tomas A. |
| Contributors | Nodwell, Justin R, Wright, Gerry, Li, Yingfu, Biochemistry |
| Source Sets | McMaster University |
| Detected Language | English |
| Type | thesis |
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