Tyurin Michael V, Desai Sunil G, Lynd Lee R
Thayer School of Engineering. Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire 03755, USA.
Appl Environ Microbiol. 2004 Feb;70(2):883-90. doi: 10.1128/AEM.70.2.883-890.2004.
Electrotransformation of several strains of Clostridium thermocellum was achieved using plasmid pIKm1 with selection based on resistance to erythromycin and lincomycin. A custom-built pulse generator was used to apply a square 10-ms pulse to an electrotransformation cuvette consisting of a modified centrifuge tube. Transformation was verified by recovery of the shuttle plasmid pIKm1 from presumptive transformants of C. thermocellum with subsequent PCR specific to the mls gene on the plasmid, as well as by retransformation of Escherichia coli. Optimization carried out with strain DSM 1313 increased transformation efficiencies from <1 to (2.2 +/- 0.5) x 10(5) transformants per micro g of plasmid DNA. Factors conducive to achieving high transformation efficiencies included optimized periods of incubation both before and after electric pulse application, chilling during cell collection and washing, subculture in the presence of isoniacin prior to electric pulse application, a custom-built cuvette embedded in an ice block during pulse application, use of a high (25-kV/cm) field strength, and induction of the mls gene before plating the cells on selective medium. The protocol and preferred conditions developed for strain DSM 1313 resulted in transformation efficiencies of (5.0 +/- 1.8) x 10(4) transformants per micro g of plasmid DNA for strain ATCC 27405 and approximately 1 x 10(3) transformants per micro g of plasmid DNA for strains DSM 4150 and 7072. Cell viability under optimal conditions was approximately 50% of that of controls not exposed to an electrical pulse. Dam methylation had a beneficial but modest (7-fold for strain ATCC 27405; 40-fold for strain DSM 1313) effect on transformation efficiency. The effect of isoniacin was also strain specific. The results reported here provide for the first time a gene transfer method functional in C. thermocellum that is suitable for molecular manipulations involving either the introduction of genes associated with foreign gene products or knockout of native genes.
利用携带基于对红霉素和林可霉素抗性筛选标记的质粒pIKm1，实现了多株热纤梭菌的电转化。使用定制的脉冲发生器向由改良离心管组成的电转化比色杯中施加10毫秒的方形脉冲。通过从热纤梭菌推定转化体中回收穿梭质粒pIKm1，随后进行针对质粒上mls基因的PCR，以及通过大肠杆菌的再转化来验证转化。对DSM 1313菌株进行的优化将转化效率从每微克质粒DNA小于1个转化体提高到(2.2±0.5)×10⁵个转化体。有助于实现高转化效率的因素包括电脉冲施加前后的优化孵育时间、细胞收集和洗涤过程中的冷却、电脉冲施加前在异烟肼存在下的传代培养、脉冲施加期间嵌入冰块中的定制比色杯、使用高(25 kV/cm)场强以及在将细胞接种到选择性培养基之前诱导mls基因。为DSM 1313菌株开发的方案和优选条件导致ATCC 27405菌株的转化效率为每微克质粒DNA(5.0±1.8)×10⁴个转化体，DSM 4150和7072菌株的转化效率约为每微克质粒DNA 1×10³个转化体。最佳条件下的细胞活力约为未接受电脉冲处理的对照细胞的50%。Dam甲基化对转化效率有有益但适度的影响(ATCC 27405菌株为7倍；DSM 1313菌株为40倍)。异烟肼的作用也是菌株特异性的。本文报道的结果首次提供了一种在热纤梭菌中起作用的基因转移方法，该方法适用于涉及引入与外源基因产物相关的基因或敲除天然基因的分子操作。
