Creating a Library of Random Transposon Mutants in Leptospira.
Creating a Library of Random Transposon Mutants in Leptospira.
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DOI：
10.1007/978-1-0716-0459-5_8
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发表时间：
2020
影响因子：
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通讯作者：
Christopher J. Pappas;Hui Xu;Md A. Motaleb
中科院分区：
文献类型：
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作者：
Christopher J. Pappas;Hui Xu;Md A. Motaleb
随机转座子突变文库的建立在钩端螺旋体的定点突变中是有利的，因为定点突变仍然是一个挑战，特别是在致病物种中。这一过程通常是通过以大肠杆菌为供体细胞，将含有Himar1的质粒转化为钩端螺旋体来完成的。在这里，我们描述了在含有pSW29T-TKS2的质粒pSW29T-TKS2的接合中产生随机转座子突变体的方法。Coliβ2163。还将描述用半随机套式聚合酶链式反应确定转座子插入位点。类似的方法也可用于产生致病钩端螺旋体的TN突变体。
Generation of a random transposon mutant library is advantageous inLeptospiraas site-directed mutagenesis remains a challenge, especially in pathogenic species. This procedure is typically completed by transformation ofLeptospirawith aHimar1containing plasmid via conjugation withEscherichia colias a donor cell. Here we describe the methodology to generate random transposon mutants in the saprophyteLeptospira biflexavia conjugation of plasmid pSW29T-TKS2 harbored inE. coliβ2163. Determination of transposon insertion site by semi-random nested PCR will also be described. A similar methodology may be employed to generate Tn mutants of pathogenicLeptospiraspecies.