An Integron-based Synthetic Genomic Platform from Vibrio vulnificus - Artificial AcMNPV Synthesis

• Main Authors: Aldrich Agtarap, 艾傑克
• Other Authors: Chung-Yung Chen
• Format: Others
• Language: en_US
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/15767406797239461796

碩士 === 中原大學 === 生物科技研究所 === 99 === Integrons are assembly platforms that acquire open reading frames, called gene cassettes, and convert them into functional genes. Gene cassettes are notable for coding antibiotic resistance genes indicating that integrons have played a major role in the emergence of multidrug-resistant bacteria. Successive integrations of gene cassettes into the integron platform result to an array of functional gene units which is apparent in Vibrio vulnificus genome, 188 gene cassettes in tandem. Here, we will assemble pieces of DNA into a bigger DNA molecule by mimicking the intrinsic gene capture system of the integron from V. vulnificus. V. vulnificus’ integron platform was cloned into a BAC vector to generate the synthetic genomic tool (SGT). A reporter gene cassette was synthesized by placing DsRed next to a recombination site, attC, followed by self-ligation. As a first step in exploiting the integron of V. vulnificus, the SGT will be the landing platform of the reporter gene cassette along with 64 synthetic AcMNPV gene cassettes resulting to an array of functional viral gene units that corresponds to AcMNPV mini-genome. AcMNPV is a rod-shaped virus and was previously engineered as an expression vector system that has been extensively used for gene expression in both insect and mammalian cells. This AcMNPV mini-genome is the truncated version of the original AcMNPV genome, from 154 to 64 ORFs. Removing unnecessary AcMNPV ORFs reduces the time (virus matures faster) and cost (lesser amounts of nutrients required during viral propagation) in the process of producing recombinant proteins. We introduced the reporter gene cassette into the SGT in several ways to initiate the assembly of AcMNPV mini-genome. 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. A similar result was observed by Biskri et al. (JB 187:1740, 2005) when Vibrio cholerae’s integron platform was reconstituted in E. coli. 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.