Date of Award
Master of Science (MSc)
Justin R Nodwell
The Gram-positive, soil dwelling bacteria of the genus Streptomyces produce greater than 50% of the clinically relevant antibiotics in use today. Thanks to the falling price of DNA sequencing, Streptomyces 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 Streptomyces coelicolor in several other Streptomyces 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 Streptomyces. With these two technologies in place, an attempt was to made to introduce three plasmids (pSET152-ermE*p-null, pSET152-ermE*p-atrA and pSET152-ermE*p-lsr2NTD) into 120 wild-isolate Streptomyces species from the Wright Actinomycete Collection. Exconjugants were successfully obtained for all three plasmids in 48 species of Streptomyces and were screened for increased antimicrobial activity using a HTP, lux-based bioassay. Numerous strains showed increased antimicrobial activity but WAC00206, WAC00230 and WAC00263 with pSET152-ermE*p-lsr2NTD 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 lux-based bioassay are powerful methods for introducing plasmids into and screening engineered streptomycetes.
Gverzdys, Tomas A., "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" (2011). Open Access Dissertations and Theses. Paper 6090.
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