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首页> 外文期刊>Applied and Environmental Microbiology >In vivo methylation in Escherichia coli by the Bacillus subtilis phage phi 3T I methyltransferase to protect plasmids from restriction upon transformation of Clostridium acetobutylicum ATCC 824.
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In vivo methylation in Escherichia coli by the Bacillus subtilis phage phi 3T I methyltransferase to protect plasmids from restriction upon transformation of Clostridium acetobutylicum ATCC 824.

机译：枯草芽孢杆菌噬菌体phi 3T I甲基转移酶在大肠杆菌中进行体内甲基化，以保护质粒不受丙酮丁醇梭菌ATCC 824转化的限制。

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摘要

The restriction endonuclease Cac824I has been shown to be a major barrier to electrotransformation of Clostridium acetobutylicum ATCC 824 (L. D. Mermelstein, N. E. Welker, G. N. Bennett, and E. T. Papoutsakis, Bio/Technology 10:190-195, 1992). Methylation by the phi 3T I methyltransferase encoded by Bacillus subtilis phage phi 3T was shown to protect plasmid DNA from restriction by Cac824I. Expression in Escherichia coli of the phi 3tI gene (which encodes the phi 3T I methyltransferase) from pAN1, which replicates via the p15A origin of replication, was sufficient to completely methylate coresident E. coli-C. acetobutylicum shuttle vectors with ColE1 origins of replication. Three shuttle vectors (pIMP1, pSYL2, and pSYL7) methylated in this manner were used to efficiently electrotransform strain ATCC 824. These vectors could not be introduced into strain ATCC 824 when unmethylated because the E. coli portions of the plasmids contain a large number of Cac824I sites. This method obviates the need to use B. subtilis-C. acetobutylicum shuttle vectors with few Cac824I sites to introduce DNA into C. acetobutylicum ATCC 824.
机译：已经显示限制性核酸内切酶Cac824I是乙酰丁酸梭菌ATCC 824的电转化的主要障碍（L.D.Mermelstein，N.E.Welker，G.N.Bennett，和E.T.Papoutsakis，Bio / Technology 10：190-195，1992）。已显示由枯草芽孢杆菌噬菌体phi 3T编码的phi 3T I甲基转移酶甲基化可保护质粒DNA不受Cac824I的限制。通过p15A复制起点复制的pAN1中的phi 3tI基因（编码phi 3T I甲基转移酶）在大肠杆菌中的表达足以完全使共存的大肠杆菌C甲基化。具有ColE1复制起点的乙草酰穿梭载体。以这种方式甲基化的三个穿梭载体（pIMP1，pSYL2和pSYL7）用于有效地电转化ATCC 824菌株。未甲基化时，这些载体不能导入ATCC 824菌株，因为质粒的大肠杆菌部分含有大量的Cac824I网站。该方法消除了使用枯草芽孢杆菌-C的需要。带有少量Cac824I位点的乙草丁穿梭载体，可将DNA引入乙草丁香ATCC 824中。

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