Construction of potential unmarked gene deletion mutagenesis systems and selection of regulable promoters used in Rhodococcus equi
| Autor: | Yu-Wen Luo, 羅昱文 |
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| Rok vydání: | 2015 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 103 Rhodococcus equi, a Gram-positive bacterium, has the ability to assimilate sterols. Despite the metabolic significance of R. equi, development of genetic engineering has been restricted by limitations inherent in the available molecular tools. To facilitate genetic manipulation of R. equi, we sought for suitable counter-selectable markers for unmarked gene deletion and regulable promoters applicable in gene expression. 3-ketosteroid 9α-monooxygenase subunit A (KSHA) encoded by REQ_40110 is a key enzyme in catabolic pathway of steroids, and its promoter is regulated by androstenedione (AD). We examined the optimal induction condition of kshA promoter, and constructed an inducible expression plasmid with the regulatory element. Several suicide genes including rpsL, sacB and pheS were characterized in R. equi. The P-rpsL cassette consisting of rpsL and its promoter was ineffective in negative selection using streptomycin because of mutations of 16S rRNA in R. equi strain USA-18ΔB8. On the other hand, the novel cassettes, PkshA-sacB and PkshA-pheS**, a mutant version of pheS with T273S and A320G double mutations, were created and conferred conditional lethality to R. equi cells. For example, PkshA-sacB cassette enables host cells sensitive to sucrose under induction of AD, and PkshA-pheS** cassette brings about cytotoxic effect in presence of p-chloro-phenylalanine and AD. PkshA-sacB and PkshA-pheS** cassettes are verified as potential counter-selectable markers for unmarked gene deletion method during preliminary experiments. Besides, a constitutive promoter was found via promoter selection using promoter-probe plasmid but it has not been defined. In summary, the findings in this study may be helpful to further genetic analysis of R. equi. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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