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http://hdl.handle.net/11375/11095
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DC Field | Value | Language |
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dc.contributor.advisor | Nodwell, Justin R | en_US |
dc.contributor.advisor | Wright, Gerry | en_US |
dc.contributor.advisor | Li, Yingfu | en_US |
dc.contributor.author | Gverzdys, Tomas A. | en_US |
dc.date.accessioned | 2014-06-18T16:53:34Z | - |
dc.date.available | 2014-06-18T16:53:34Z | - |
dc.date.created | 2011-08-30 | en_US |
dc.date.issued | 2011-10 | en_US |
dc.identifier.other | opendissertations/6090 | en_US |
dc.identifier.other | 7116 | en_US |
dc.identifier.other | 2205330 | en_US |
dc.identifier.uri | http://hdl.handle.net/11375/11095 | - |
dc.description.abstract | <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> | en_US |
dc.subject | luminescence | en_US |
dc.subject | lux | en_US |
dc.subject | conjugation | en_US |
dc.subject | screening | en_US |
dc.subject | HTP | en_US |
dc.subject | HTS | en_US |
dc.subject | Biochemistry | en_US |
dc.subject | Biotechnology | en_US |
dc.subject | Biochemistry | en_US |
dc.title | The Development of Protocols to Engineer and Screen Streptomyces in High Throughput to Test for the Activation of Cryptic Clusters by the Heterologous Expression of Pleiotropic Regulators | en_US |
dc.type | thesis | en_US |
dc.contributor.department | Biochemistry | en_US |
dc.description.degree | Master of Science (MSc) | en_US |
Appears in Collections: | Open Access Dissertations and Theses |
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File | Size | Format | |
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fulltext.pdf | 5.3 MB | Adobe PDF | View/Open |
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