The gene knockouts are mostly created using homologous recombination-based replacement of target gene(s) with the expressing cassette of selection marker gene(s). Here, we constructed a series of plasmids bearing the expressing cassettes of genes encoding different antibiotics markers like nptII (KanR), aadA (SpecR), cat (CmR) and aac(3) (GenR). D. radiodurans is a radioresistant Gram positive bacterium that does not support the independent maintenance of colE1 origin-based plasmids. Using these constructs, the disruption mutants of both single and multiple genes involved in segregation of secondary genome elements have been generated in this bacterium. Unlike single mutants, the double and triple mutants showed growth retardation under normal growth conditions and the synergistic effects with Topoisomerase II inhibitor on the growth of this bacterium. Thus, these plasmids could be useful in creating multiple deletions/disruptions in bacteria that do not support independent maintenance of colE1 origin-based plasmid.

中文翻译：

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用于在耐辐射细菌Deinococcus radiodurans中进行多次敲除的质粒。

基因敲除主要是通过用选择标记基因的表达盒对靶基因进行基于同源重组的替换而产生的。在这里，我们构建了一系列带有编码不同抗生素标记（例如nptII（KanR），aadA（SpecR），cat（CmR）和aac（3）（GenR））的基因表达盒的质粒。D. radiodurans是一种耐辐射的革兰氏阳性细菌，不支持基于colE1起源质粒的独立维护。使用这些构建体，已经在该细菌中产生了与次级基因组元件的分离有关的单个和多个基因的破坏突变体。与单突变体不同 双突变体和三突变体在正常生长条件下显示出生长迟缓，并且与拓扑异构酶II抑制剂对该细菌的生长具有协同作用。因此，这些质粒可用于在不支持基于colE1起源质粒的独立维护的细菌中产生多个缺失/破坏。