van Randen J, Venema G
Mol Gen Genet. 1984;195(1-2):57-61. doi: 10.1007/BF00332724.
We have cloned the hisH tyrA wild-type genes of Bacillus subtilis with the aid of the chimeric plasmid pBJ194, which replicates both in B. subtilis and Escherichia coli. Primary cloning was done in E. coli. The original E. coli clone, carrying the recombinant plasmid (pGR1) which complements hisH tyrA mutants of B. subtilis, was selected directly from a mixture of plated E. coli clones by replicaplating these clones onto minimal agar plates without tyrosine, spread just before with competent B. subtilis cells. After overnight incubation clusters of small colonies had developed exclusively in the E. coli [pGR1] colony prints. The Tyr+ minicolonies were shown to be B. subtilis carrying pGR1 because (i) their appearance depended linearly on the number of B. subtilis cells plated, (ii) they produced extracellular protease and amylase and (iii) plasmids could be reisolated from the minicolonies and used to transform B. subtilis recE4 tyrA1 both to Cmr and Tyr+. Plasmid pGR1 transfer through replica plating was compared with plasmid transfer in liquid. Both systems depended on transformable B. subtilis strains and were sensitive to DNAseI. However, whereas integration of the tyrA+ gene into the chromosome and concomitant loss of plasmids occurred frequently during regular plasmid transformation of Rec+ B. subtilis, this was a rare event during plasmid transfer through replica plating.
我们借助嵌合质粒pBJ194克隆了枯草芽孢杆菌的hisH tyrA野生型基因，该质粒可在枯草芽孢杆菌和大肠杆菌中复制。初次克隆在大肠杆菌中进行。通过将平板培养的大肠杆菌克隆复制到不含酪氨酸的基本琼脂平板上，直接从平板培养的大肠杆菌克隆混合物中筛选出携带能互补枯草芽孢杆菌hisH tyrA突变体的重组质粒（pGR1）的原始大肠杆菌克隆，就在铺板前用感受态枯草芽孢杆菌细胞进行涂布。过夜培养后，仅在大肠杆菌[pGR1]菌落印记中出现了小菌落簇。已证明Tyr +小菌落是携带pGR1的枯草芽孢杆菌，因为（i）它们的出现与所铺枯草芽孢杆菌细胞的数量呈线性关系，（ii）它们产生细胞外蛋白酶和淀粉酶，并且（iii）质粒可从小菌落中重新分离出来，并用于将枯草芽孢杆菌recE4 tyrA1转化为Cmr和Tyr +。将通过复制平板法进行的质粒pGR1转移与液体中的质粒转移进行了比较。两种系统都依赖于可转化的枯草芽孢杆菌菌株，并且对DNAseI敏感。然而，在Rec +枯草芽孢杆菌的常规质粒转化过程中，tyrA +基因经常整合到染色体中并伴随质粒丢失，而在通过复制平板法进行的质粒转移过程中，这种情况很少发生。
