Kumar Ashok, Bag Satyabrata, Das Bhabatosh
Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, India.
School of Life Sciences, Manipal University, Manipal-576104, Karnataka, India.
Recent Pat Biotechnol. 2018;12(3):200-207. doi: 10.2174/1872208312666180223113618.
Genomic islands (GIs) are discrete segments of mobile DNA with defined boundaries according to recent patents, acquired in the bacterial genome from another organism by horizontal gene transfer during the course of evolution. GIs contribute significantly to virulence, disease development, antimicrobial resistance and metabolic process.
The present study focuses on the development of a vector based genetic tool carrying selectable and counter-selectable markers, in order to flag the GIs in the bacterial chromosome and monitor their stability under in vitro and in vivo conditions.
We engineered suicide vectors, pSB40 and pSB41, carrying single or tandem copies of chloramphenicol acetyltransferase (cat) and levansucrase (sacB) alleles, respectively. The sacB-cat allele in both the vectors is flanked by several restriction sites. To test the suitability of sacB-cat allele for monitoring GI loss, we introduced the allele in the Vibrio Pathogenicity Island-1 (VPI-1) in Vibrio cholerae genome.
The V. cholerae strain carrying sacB-cat allele in VPI-1 element showed resistance to chloramphenicol and sensitivity to sucrose at optimal growth conditions. Loss of VPI-1 element from the V. cholerae genome was simply monitored by growing the cells on selection agar plates supplemented with sucrose. Our results showed that the genetic tool we developed is suitable for monitoring GI stability in the bacterial genome.
The present study indicates that pSB40 and pSB41are efficient and sensitive genetic tool that can be used for reverse genetics experiments and monitoring stability of mobile genetic elements in the bacterial genome.
根据最近的专利，基因组岛（GIs）是具有明确边界的可移动DNA离散片段，在进化过程中通过水平基因转移从另一种生物体获得并整合到细菌基因组中。基因组岛对细菌的毒力、疾病发展、抗菌抗性和代谢过程有重要贡献。
本研究重点开发一种基于载体的遗传工具，该工具带有可选择和反选择标记，以便标记细菌染色体中的基因组岛，并监测其在体外和体内条件下的稳定性。
我们构建了自杀载体pSB40和pSB41，分别携带氯霉素乙酰转移酶（cat）和果聚糖蔗糖酶（sacB）等位基因的单拷贝或串联拷贝。两个载体中的sacB-cat等位基因两侧有多个限制性酶切位点。为了测试sacB-cat等位基因用于监测基因组岛丢失的适用性，我们将该等位基因引入霍乱弧菌基因组中的霍乱弧菌致病岛-1（VPI-1）。
在VPI-1元件中携带sacB-cat等位基因的霍乱弧菌菌株在最佳生长条件下对氯霉素具有抗性，对蔗糖敏感。通过在补充有蔗糖的选择琼脂平板上培养细胞，可简单监测霍乱弧菌基因组中VPI-1元件的丢失。我们的结果表明，我们开发的遗传工具适用于监测细菌基因组中基因组岛的稳定性。
本研究表明，pSB40和pSB41是高效且灵敏的遗传工具，可用于反向遗传学实验和监测细菌基因组中可移动遗传元件的稳定性。
