A novel synthetic genomic tool from Vibrio vulnificus CG021--antibiotic synthesis

• Main Authors: Chen-Chang Yang, 楊鎮璋
• Other Authors: Chung-Yung Chen
• Format: Others
• Language: en_US
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/92650602748339411201

碩士 === 中原大學 === 生物科技研究所 === 99 === Amphotericin B is a polyene antibiotic active against fungi. Availability of amphotericin B is limited by the amounts produced by Streptomyces nodosus. Here, we will assemble amphotericin B genes as gene cassettes using the intrinsic gene-capture system of integrons. Integrons are platforms that assemble open reading frames in a form of gene cassettes and convert them into functional genes. The isolated integron platform from Vibrio vulnificus CG021 was inserted into a BAC vector to construct the synthetic genomic tool (SGT). A reporter gene cassette was synthesized by placing DsRed next to a recombination site followed by self-ligation. As a first step in exploiting the integron platform of V. vulnificus, the SGT will be the landing platform of the reporter gene cassette along with amphotericin B gene cassettes in Escherichia coli. The expression of assembled amphotericin B gene cassettes will be analyzed by Q-PCR. We introduced the reporter gene cassette into the SGT in several ways to initiate the assembly of amphotericin B genes. Thousands of colonies were screened but the SGT was found empty. In this study, the reconstituted integron platform in E. coli was found non-functional. One possible explanation for the observation is the absence or incompatibility of a species-specific accessory protein sustaining the integrase-mediated integration process. Our result suggests that the integration of gene cassettes into the integron platform might be more complex than previously proposed.