Comparing insertion libraries in two Pseudomonas aeruginosa strains to assess gene essentiality.
Comparing insertion libraries in two Pseudomonas aeruginosa strains to assess gene essentiality.
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比较两种铜绿假单胞菌菌株中的插入文库以评估基因必要性。
DOI：
10.1007/978-1-59745-321-9_10
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发表时间：
2008
期刊：
影响因子：
--
通讯作者：
Ausubel,FrederickM
中科院分区：
文献类型：
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作者：
Liberati,NicoleT;Urbach,JonathanM;Thurber,TaraK;Wu,Gang;Ausubel,FrederickM
假定的必要基因可以通过比较相同细菌种类的相关菌株中多个近饱和转座子插入突变文库中未中断的同源物来鉴定。描述了鉴定两种细菌菌株之间所有同源物和假定的基本同源物的方法。此外，详细介绍了细菌近饱和转座子插入诱变的方案，包括(1)偶联介导的诱变，(2)突变培养物的自动集落采摘和液体处理，以及(3)任意聚合酶链反应扩增和转座子插入位点附近基因组DNA测序。
Putative essential genes can be identified by comparing orthologs not disrupted in multiple near-saturated transposon insertion mutation libraries in related strains of the same bacterial species. Methods for identifying all orthologs between two bacterial strains and putative essential orthologs are described. In addition, protocols detailing near-saturation transposon insertion mutagenesis of bacteria are presented, including (1) conjugation-mediated mutagenesis, (2) automated colony picking and liquid handling of mutant cultures, and (3) arbitrary polymerase chain reaction amplification and sequencing of genomic DNA adjacent to transposon insertion sites.