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首页> 外文期刊>Plasmid: An International Journal Devoted to Extrachromosomal Gene Systems >Modified RP4 and Tn5-Mob derivatives for facilitated manipulation of large plasmids in Gram-negative bacteria.
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Modified RP4 and Tn5-Mob derivatives for facilitated manipulation of large plasmids in Gram-negative bacteria.

机译：修饰的RP4和Tn5-Mob衍生物，可促进革兰氏阴性细菌中大质粒的操作。

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摘要

We have constructed a set of RP4 (NmS/TcS) and Tn5-Mob derivatives which have applications in experiments involving mobilization of replicons in many Gram-negative organisms. The different selection markers of the RP4 and Tn5-Mob derivatives include streptomycin, chloramphenicol, gentamicin, and spectinomycin resistance as well as mercury resistance, and a constitutively expressed lacZ gene. This choice of markers allows the use of these derivatives in bacteria which are naturally resistant to many antibiotics, and in strains which contain pre-existing resistance plasmids, transposons, or antibiotic cassette insertions. In addition, a RP4 derivative carrying the sacB gene of Bacillus subtilis was constructed. This allows the selection for the loss of RP4 after it has been used to mobilize other plasmids. A Tn5-Mob-sacB derivative with a new marker (Gm) was also developed, as were vectors which take advantage of the sacB gene to facilitate replacement of existing Tn5 inserts with other Tn5 derivatives. As an example of the use of these tools, three Rhizobium leguminosarum bv. viciae VF39 plasmids which have been shown to be involved in symbiosis were differentially tagged and mobilized (individually and in various combinations) to the plasmid-free Agrobacterium tumefaciens strain UBAPF2. None of the resultant Agrobacterium strains was able to fix nitrogen in symbiosis with peas.
机译：我们构建了一套RP4（NmS / TcS）和Tn5-Mob衍生物，它们在涉及动员许多革兰氏阴性生物中的复制子的实验中具有应用。 RP4和Tn5-Mob衍生物的不同选择标记包括链霉素，氯霉素，庆大霉素和壮观霉素的抗性以及耐汞性，以及组成型表达的lacZ基因。这种标记的选择允许将这些衍生物用于对许多抗生素具有天然抗性的细菌中，以及含有预先存在的抗性质粒，转座子或抗生素盒插入物的菌株中。另外，构建了携带枯草芽孢杆菌sacB基因的RP4衍生物。在用于动员其他质粒后，可以选择RP4的缺失。还开发了具有新标记（Gm）的Tn5-Mob-sacB衍生物，以及利用sacB基因来促进其他Tn5衍生物替换现有Tn5插入片段的载体。以使用这些工具为例，介绍了三个豆科根瘤菌。将已显示参与共生的蚕豆VF39质粒进行差异标记，并分别（分别和以各种组合）动员至无质粒的根癌农杆菌菌株UBAPF2。所得的农杆菌菌株均不能在与豌豆共生的过程中固氮。

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