State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
Lett Appl Microbiol. 2012 Jul;55(1):73-81. doi: 10.1111/j.1472-765X.2012.03263.x. Epub 2012 May 25.
The aims of this study were to create and evaluate the Gateway-compatible plasmids for investigating the function of genes in Vibrio alginolyticus and other Gram-negative bacteria.
In this study, Gateway-compatible plasmids were successfully constructed for rapid and comprehensive function analysis of genes. Taking advantage of these plasmids, the in-frame deletion mutant strains and their complemented strains of five T6SS genes, including dotU1, VEPGS_0008, VEPGS_0011, hcp2 and ppkA2, were obtained. The results illustrated that all the mutant strains showed no significant effects on extracellular protease production, expression of Hcp1, and biofilm formation when compared to the wild-type strain, but in-frame deletion of VEPGS_0008 resulted in obvious biofilm reduction and the complemented strain restored to the level of the wild-type strain. Besides, in-frame deletion of dotU1, VEPGS_0008 and ppkA2 abolished the swarming ability.
A set of Gateway-compatible vectors for internal insertion, in-frame deletion and complementation of the target genes is constructed to facilitate the general and rapid function analysis of genes involved in T6SS in Vibrio alginolyticus.
The modified Gateway-compatible plasmids greatly facilitate the high-throughput and convenient function analysis of the unidentified genes.
本研究旨在构建并评估适用于弧菌属（Vibrio）和其他革兰氏阴性菌中基因功能研究的 Gateway 兼容型质粒。
本研究成功构建了适用于基因快速综合功能分析的 Gateway 兼容型质粒。利用这些质粒，获得了 5 个 T6SS 基因（dotU1、VEPGS_0008、VEPGS_0011、hcp2 和 ppkA2）的框内缺失突变株及其互补株。结果表明，与野生型菌株相比，所有突变株在胞外蛋白酶产生、Hcp1 表达和生物膜形成方面均无显著影响，但 VEPGS_0008 的框内缺失导致生物膜明显减少，而互补株则恢复到野生型菌株的水平。此外，dotU1、VEPGS_0008 和 ppkA2 的框内缺失消除了 swarm 能力。
构建了一套适用于弧菌属中 T6SS 相关基因的内部插入、框内缺失和互补的 Gateway 兼容型载体，方便了 T6SS 相关基因的快速功能分析。
经改良的 Gateway 兼容型质粒极大地促进了对未知基因的高通量和便捷功能分析。
