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首页> 外文期刊>Infection and immunity >Construction of stable cloning vectors that do not segregate from a human fecal Escherichia coli strain in the streptomycin-treated mouse large intestine.
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Construction of stable cloning vectors that do not segregate from a human fecal Escherichia coli strain in the streptomycin-treated mouse large intestine.

机译：在链霉素处理的小鼠大肠中构建不与人粪便大肠杆菌分离的稳定克隆载体。

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摘要

Escherichia coli F-18 Col- was previously shown to be a poor colonizer of the streptomycin-treated mouse large intestine, relative to its parent, E. coli F-18. Prior to attempting to clone genes responsible for the colonization phenotype of E. coli F-18 into E. coli F-18 Col-, a suitable cloning vector had to be found. In this investigation, we report that the commonly used cloning vectors pBR322, pHC79, and pBR329 all segregate from E. coli F-18 Col- both when grown in L broth under conditions of nonselection (i.e., in vitro) and when fed to streptomycin-treated mice (i.e., in vivo). Insertion of the cer region (which promotes resolution of replicating plasmids into monomeric forms) into pHC79 stabilized this plasmid in E. coli F-18 Col- in vitro and in vivo. In contrast, two independent cer insertions into pBR329 did not stabilize the plasmid completely in E. coli F-18 Col- in vitro, and feeding the strain to streptomycin-treated mice resulted in rapid segregation of the plasmids in vivo. Also, stability of all three plasmids in E. coli F-18 Col- in vitro was achieved by insertion of the parB region of plasmid R1, which encodes a cell-killing protein, Hok, that is active only postsegregationally. However, as with cer, complete in vitro and in vivo stabilization was achieved only in parB constructs of pBR322 and pHC79.
机译：相对于其亲本大肠杆菌F-18，以前已证明大肠杆菌F-18 Col-是链霉素处理过的小鼠大肠的弱定居者。在尝试将负责大肠杆菌F-18定殖表型的基因克隆到大肠杆菌F-18 Col-之前，必须找到合适的克隆载体。在这项研究中，我们报告了当在非选择条件下（即体外）在L肉汤中生长和喂入链霉素时，常用的克隆载体pBR322，pHC79和pBR329均从大肠杆菌F-18 Col-中分离出来。 -治疗的小鼠（即体内）。在大肠杆菌F-18 Col中，在体外和体内将cer区域插入cer（促进复制质粒分解为单体形式的解析），使其稳定在大肠杆菌F-18 Col中。相比之下，在pBR329中两次独立的cer插入不能在体外在大肠杆菌F-18 Col中完全稳定该质粒，并且将该菌株喂食经链霉素处理的小鼠会导致体内质粒的快速分离。同样，通过插入质粒R1的parB区实现了这三个质粒在大肠杆菌F-18 Col中的稳定性，R1的parB区编码仅在分离后才有活性的细胞杀伤蛋白Hok。但是，与cer一样，仅在pBR322和pHC79的parB构建体中才能实现完全的体外和体内稳定。

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