Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/90040
標題: Construction of potential unmarked gene deletion mutagenesis systems and selection of regulable promoters used in Rhodococcus equi
建立適合馬紅球菌之具潛力非標記基因刪除系統及篩選其可調控啟動子
作者: 羅昱文
Yu-Wen Luo
關鍵字: 馬紅球菌
非標記基因刪除
反向篩選標誌
可調控啟動子
Rhodococcus equi
unmarked gene deletion
counterselectable markers
regulable promoters
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摘要: 馬紅球菌 (Rhodococcus equi) 屬於革蘭氏陽性菌,具有良好的固醇分解能力。儘管馬紅球菌在固醇代謝上的重要性,其基因工程之發展一直受限於缺乏有效的分子工具。為了能夠促進基因體學上之操作,本實驗的目標為尋找可用於非標記基因刪除系統的反向篩選標誌,以及可受調控的啟動子。由馬紅球菌之基因REQ_40110所表達 3-ketosteroid 9α-monooxygenase subunit A (KSHA)為固醇代謝途徑中之關鍵酵素,此蛋白可受到雄二烯酮。我們將kshA之啟動子進行檢測,並優化其誘導條件,接著以此做為調控元件而構築基因表達載體。本實驗測試了多個自殺基因的特性,包含rpsL、sacB和pheS ,並瞭解其在馬紅球菌中是否具有篩選效果。由rpsL和其自身啟動子所組成的P-rpsL基因卡匣,無法對馬紅球菌之USA-18ΔB8系列菌株進行反向篩選,原因為其16S rRNA上之突變點可能影響了菌株對鏈黴素的敏感性。另一方面,我們將另外兩個自殺基因分別和kshA啟動子結合,創造了新型的基因卡匣PkshA-sacB和具有T273S和A320G突變點的PkshA-pheS**,而其在特定條件下皆會造成寄主細胞死亡。比如說在雄二烯酮的誘導下,PkshA-sacB基因卡匣會使得寄主細胞對蔗糖具有敏感性;而當同時存在雄二烯酮和p-chloro-phenylalanine時,PkshA-pheS**基因卡匣則會引起馬紅球菌之細胞毒性效應。經過初步的實驗證實,PkshA-sacB和PkshA-pheS**基因卡匣有機會能夠成為反向篩選標誌。此外,利用啟動子偵測載體而篩選到了一個可能為常態表達的啟動子,但是其身分仍然未確定。總而言之,這些分生工具將會有助於往後對於馬紅球菌的基因體研究。
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.
URI: http://hdl.handle.net/11455/90040
文章公開時間: 2016-08-27
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