Onai K, Morishita M, Kaneko T, Tabata S, Ishiura M
Center for Gene Research, Nagoya University, Furo, Chikusa, 464-8602 Nagoya, Japan.
Mol Genet Genomics. 2004 Feb;271(1):50-9. doi: 10.1007/s00438-003-0953-9. Epub 2003 Nov 25.
Proteins derived from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1, which performs plant-type oxygenic photosynthesis, are suitable for biochemical, biophysical and X-ray crystallographic studies. We found that T. elongatus displays natural transformation, and we established a simple and efficient protocol for transferring exogenous DNAs into the organism's genome. We obtained transformants directly on selective agar plates without having to amplify them prior to plating. We constructed several targeting vectors that enabled us to insert exogenous DNAs into specific sites without disrupting endogenous genes and operons. We also developed a new selectable marker gene for T. elongatus by optimizing the codons of the gene encoding a kanamycin nucleotidyltransferase derived from the thermophilic bacterium Bacillus stearothermophilus. This synthetic gene enabled us to select transformants as kanamycin-resistant colonies on agar plates at 52 degrees C. Optimization of the conditions for natural transformation resulted in a transformation efficiency of up to 1.7 x 10(3) transformants per microg of DNA. The exogenous DNAs were integrated stably into the targeted sites of the T. elongatus genome via homologous recombination by double crossovers.
来源于嗜热蓝细菌嗜热栖热放线菌BP-1（其进行植物型产氧光合作用）的蛋白质，适用于生化、生物物理和X射线晶体学研究。我们发现嗜热栖热放线菌具有自然转化能力，并且我们建立了一种简单有效的方法，用于将外源DNA转移到该生物体的基因组中。我们直接在选择性琼脂平板上获得了转化体，而无需在平板接种前对其进行扩增。我们构建了几种靶向载体，使我们能够将外源DNA插入特定位点而不破坏内源基因和操纵子。我们还通过优化来源于嗜热芽孢杆菌的卡那霉素核苷酸转移酶编码基因的密码子，为嗜热栖热放线菌开发了一种新的选择标记基因。这个合成基因使我们能够在52℃的琼脂平板上选择作为卡那霉素抗性菌落的转化体。自然转化条件的优化导致转化效率高达每微克DNA 1.7×10³个转化体。外源DNA通过双交换同源重组稳定地整合到嗜热栖热放线菌基因组的靶向位点。
