A rapid seamless method for gene knockout in Pseudomonas aeruginosa.
A rapid seamless method for gene knockout in Pseudomonas aeruginosa.
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DOI：
10.1186/s12866-017-1112-5
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发表时间：
2017-09-19
期刊：
影响因子：
4.2
通讯作者：
Wilks A
中科院分区：
文献类型：
--
作者：
Huang W;Wilks A
铜绿假单胞菌是研究群体感应、生物膜形成的模式生物，也是免疫功能低下患者医院感染的主要原因。因此，铜绿假单胞菌在其遗传学方面是研究最充分的生物体之一。然而，在铜绿假单胞菌中构建基因缺失和置换相对耗时，需要多个步骤，包括自杀载体构建、缀合、插入抗生素抗性盒的失活和等位基因交换。即使采用Gateway重组工程技术进行直接转化，也需要至少两周的时间。我们已经开发了一种快速简化的方法，通过直接转化在铜绿假单胞菌中产生干净的缺失突变体，消除了创建Gateway兼容自杀载体的需要。在该方法中，通过聚合酶链式反应（PCR）扩增待缺失的基因/基因座的上游和下游序列，并通过吉布森组装与线性化的pEX 18 Tc sacB自杀质粒无缝融合。通过本研究中优化的电穿孔方法将所得缺失质粒转化到铜绿假单胞菌中。然后通过同源重组将质粒整合到染色体中，并通过sacB介导的蔗糖反选择鉴定缺失突变体。本方法用于产生血红素同化系统抗σ因子hasS和分别涉及ECF系统抗σ和σ因子vreA和vreI的毒力调节因子的干净基因缺失。从质粒构建到通过DNA测序确认基因缺失的过程在一周内完成。此外，该方法的实用性在构建vreA和vreI缺失中突出显示，其中vreA的起始密码子和vreI的终止密码子重叠。利用吉布森组装缺失突变体，以单碱基对精确度构建，以产生相应的vreA和vreI缺失，同时保持相应基因的起始和终止密码子。总的来说，该方法允许以碱基对精确度快速构建铜绿假单胞菌中的基因缺失。这种方法从自杀载体的构建到未标记基因缺失的序列确认可以在一周内进行，而不需要昂贵的专有试剂或仪器。吉布森组装的精确度和产生所需构建体的准确度为95%的事实，使其成为先前方法的可行且有吸引力的替代方案。本文的在线版本（10.1186/s12866-017-1112-5）包含补充材料，可供授权用户使用。
Pseudomonas aeruginosa is a model organism for the study of quorum sensing, biofilm formation, and also leading cause of nosocomial infections in immune compromised patients. As such P. aeruginosa is one of the most well studied organisms in terms of its genetics. However, the construction of gene deletions and replacements in Pseudomonas aeruginosa is relatively time-consuming, requiring multiple steps including suicide vector construction, conjugation, inactivation with insertion of antibiotic resistance cassettes and allelic exchange. Even employing Gateway recombineering techniques with direct transformation requires a minimum two weeks. We have developed a rapid streamlined method to create clean deletion mutants in P. aeruginosa through direct transformation, eliminating the need for the creation of Gateway-compatible suicide vectors. In this method, upstream and downstream sequences of the gene/locus to be deleted are amplified by polymerase chain reaction (PCR) and seamlessly fused with the linearized pEX18Tc sacB suicide plasmid by Gibson assembly. The resulting deletion plasmid is transformed into P. aeruginosa by an electroporation method optimized in this study. The plasmid is then integrated into the chromosome by homologous recombination, and deletion mutants are identified via sacB mediated sucrose counter-selection. The current method was employed to generate clean gene deletions of the heme assimilation system anti-σ factor, hasS and the virulence regulator involving ECF system anti-σ and σ factors vreA and vreI, respectively. The process from plasmid construction to confirmation by DNA sequencing of the gene deletion was completed in one week. Furthermore, the utility of the method is highlighted in the construction of the vreA and vreI deletions, where the start codon of vreA and the stop codon of vreI overlap. Utilizing Gibson assembly deletion mutants were constructed with single base pair precision to generate the respective vreA and vreI deletions, while maintaining the start and stop codon of the respective genes. Overall, this method allows for rapid construction of gene deletions in P. aeruginosa with base pair precision. This method from the construction of the suicide vector to sequence confirmation of the unmarked gene deletion can be performed in one week, without the requirement for expensive proprietary reagents or instruments. The precision of Gibson assembly and the fact the accuracy in generating the desirable construct is 95%, makes this a viable and attractive alternative to previous methods. The online version of this article (10.1186/s12866-017-1112-5) contains supplementary material, which is available to authorized users.
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影响因子：
3.6
作者：
BLOMFIELD, IC;VAUGHN, V;EISENSTEIN, BI
通讯作者：
EISENSTEIN, BI
影响因子：
14.8
作者：
Hmelo LR;Borlee BR;Almblad H;Love ME;Randall TE;Tseng BS;Lin C;Irie Y;Storek KM;Yang JJ;Siehnel RJ;Howell PL;Singh PK;Tolker-Nielsen T;Parsek MR;Schweizer HP;Harrison JJ
通讯作者：
Harrison JJ
DOI：
10.1099/00221287-13-3-572
发表时间：
1955-01-01
期刊：
JOURNAL OF GENERAL MICROBIOLOGY
影响因子：
--
作者：
HOLLOWAY, BW
通讯作者：
HOLLOWAY, BW
影响因子：
48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.
影响因子：
11.8
作者：
Wolfgang, MC;Lee, VT;Lory, S
通讯作者：
Lory, S