Shark K B, Smith F D, Harpending P R, Rasmussen J L, Sanford J C
Department of Horticultural Sciences, Cornell University, Geneva, New York 14456.
Appl Environ Microbiol. 1991 Feb;57(2):480-5. doi: 10.1128/aem.57.2.480-485.1991.
We present a simple and rapid method for introducing exogenous DNA into a bacterium, Bacillus megaterium, utilizing the recently developed biolistic process. A suspension of B. megaterium was spread onto the surface of nonselective medium. Plasmid pUB110 DNA, which contains a gene that confers kanamycin resistance, was precipitated onto tungsten particles. Using a biolistic propulsion system, the coated particles were accelerated at high velocities into the B. megaterium recipient cells. Selection was done by use of an agar overlay containing 50 micrograms of kanamycin per ml. Antibiotic-resistant transformants were recovered from the medium interface after 72 h of incubation, and the recipient strain was shown to contain the delivered plasmid by agarose gel electrophoresis of isolated plasmid DNA. All strains of B. megaterium tested were successfully transformed by this method, although transformation efficiency varied among strains. Physical variables of the biolistic process and biological variables associated with the target cells were optimized, yielding greater than 10(4) transformants per treated plate. This is the first report of the biolistic transformation of a procaryote.
我们展示了一种利用最近开发的生物枪技术将外源DNA导入巨大芽孢杆菌的简单快速方法。将巨大芽孢杆菌的悬浮液铺展在非选择性培养基表面。含有赋予卡那霉素抗性基因的质粒pUB110 DNA沉淀在钨颗粒上。使用生物枪推进系统，将包被的颗粒以高速加速进入巨大芽孢杆菌受体细胞。通过使用每毫升含有50微克卡那霉素的琼脂覆盖物进行筛选。孵育72小时后，从培养基界面回收抗生素抗性转化体，通过对分离的质粒DNA进行琼脂糖凝胶电泳显示受体菌株含有导入的质粒。尽管不同菌株的转化效率有所不同，但所有测试的巨大芽孢杆菌菌株都通过该方法成功转化。对生物枪技术的物理变量和与靶细胞相关的生物学变量进行了优化，每处理平板产生超过10⁴个转化体。这是原核生物生物枪转化的首次报道。
