Functional identification of clt of Streptomyces linear plasmid SLP2 in conjugation.

• Main Authors: Jin-Xian Chen, 陳錦憲
• Other Authors: Chin-Hung Huang
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/esfvqh

碩士 === 國立臺北科技大學 === 化學工程與生物科技系生化與生醫工程碩士班 === 105 === Streptomyces species are Gram-positive bacteria that play a key role on decompositim in the soil ecology. The most unique feature is that they can have both circular and linear plasmids. Most of these plasmids acquire the ability of conjugal transfer. Previously elucidated conjugation mechanisms of F plasmid from E.coli which have demonstrated a site-specific single stranded cleavage (or nicking) by a plasmid-encoded TraI protein on the cis-acting origin of transfer (oriT) as a prerequisite for DNA transfer and follow by unidirectional transfer of single stranded DNA from the donor to the recipient. Conjugation mechanisms between Streptomyces and E.coli were significantly different. First, Streptomyces conjugation is based on double- stranded DNA rather than a single-stranded DNA. Second, time of occurrence is between two systems different, so currently we still can not define oriT for conjugation. The current study can only define a necessary DNA fragments (clt) that shown in cis function during conjugal event. Therefore, this study would like to know whether the starting point of Streptomyces linear plasmid conjugation transfer was clt. The strategy of this study is to insert clt into different positions of the chromosome. With the presence of SLP2, observing the relationship between the position of clt and the odds of different chromosomal chromosomal markers from donor appears to the recipient. From there we could try to conclude Streptomyces clt is oriT. The experi- mental data are different from the predicted results. Most of the offspring were donor with clt inserted rather than recipient or recombinant strains. This made to direct very- fycation recombinant event in offspring strains impossible. Furthermore, the recom- binant rate was 10-3 when the chromosome was not clt inserted.The disappearance of the recipient strain after clt insertion into SLP2 was never been reported.This study provides proposals to follow-up study.