Metzger M, Bellemann P, Schwartz T, Geider K
Max-Planck-Institut für medizinische Forschung, Abteilung Biophysik, Heidelberg, Germany.
Nucleic Acids Res. 1992 May 11;20(9):2265-70. doi: 10.1093/nar/20.9.2265.
The suicide plasmid pfdA31-Tn5 was constructed to mutagenize Erwinia amylovora and Escherichia coli strains by electorporation. This vector carries the bacteriophage fd replication origin, a beta-lactamase gene and the transposon Tn5. For propagation the plasmid depends on host cells producing fd gene-2 protein. Electroporation of E.amylovora or E.coli cells with plasmid pfdA31-Tn5 yielded more than 10(4) transposition events per micrograms DNA. We have produced and characterized transposon mutants of E.amylovora affecting either galactose metabolism or the synthesis of the phytotoxin (L)-2,5-dihydrophenylalanine. A Tn5-insertion in a gene, involved in exopolysaccharide synthesis of E.amylovora strain Ea7/74, was subcloned into vector pfdA31 and used to mutagenize E.amylovora strain Ea1/79 by site-directed recombination.
自杀质粒pfdA31-Tn5构建用于通过电穿孔法诱变梨火疫病菌和大肠杆菌菌株。该载体携带噬菌体fd复制起点、一个β-内酰胺酶基因和转座子Tn5。质粒的增殖依赖于产生fd基因-2蛋白的宿主细胞。用质粒pfdA31-Tn5对梨火疫病菌或大肠杆菌细胞进行电穿孔，每微克DNA产生超过10⁴次转座事件。我们已产生并鉴定了影响半乳糖代谢或植物毒素（L）-2,5-二氢苯丙氨酸合成的梨火疫病菌转座子突变体。一个参与梨火疫病菌Ea7/74菌株胞外多糖合成的基因中的Tn5插入片段被亚克隆到载体pfdA31中，并用于通过定点重组诱变梨火疫病菌Ea1/79菌株。
