An improved method for rapid generation of unmarked Pseudomonas aeruginosa deletion mutants.
An improved method for rapid generation of unmarked Pseudomonas aeruginosa deletion mutants.
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一种改进的方法，用于快速生成未标记的铜绿假单胞菌缺失突变体。
DOI：
10.1186/1471-2180-5-30
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发表时间：
2005-05-23
期刊：
影响因子：
4.2
通讯作者：
Schweizer, HP
中科院分区：
文献类型：
--
作者：
Choi, KH;Schweizer, HP
传统的基因替换程序仍然很耗时。它们通常需要克隆待突变的基因，用抗生素抗性盒插入灭活基因，并将质粒携带的突变等位基因与细菌染色体交换。聚合酶链反应和重组技术可以大大加快几乎所有步骤涉及基因替换过程。我们描述了一种快速生成无标记铜绿假单胞菌缺失突变体的方法。三个部分重叠的DNA片段被扩增，然后通过重叠延伸PCR拼接在一起。将得到的DNA片段体外克隆到Gateway载体pDONR221中，然后重组到Gateway兼容的基因替代载体pEX18ApGW中。质粒携带的缺失随后通过同源重组转移到铜绿假单胞菌染色体上。通过flp介导的抗生素耐药性标记的切除，最终获得无标记的缺失突变体。该方法用于删除25个编码GntR家族转录调节因子的铜绿假单胞菌基因。虽然保持了传统基因替代程序的关键特征，例如，自杀递送载体，抗生素抗性选择和蔗糖反选择，但由于该过程中涉及的一些关键步骤的流线型，特别是质粒携带的突变等位基因的构建及其向目标宿主的转移，这里描述的方法要快得多。经过适当的修改，该方法应适用于其他细菌。
Traditional gene replacement procedures are still time-consuming. They usually necessitate cloning of the gene to be mutated, insertional inactivation of the gene with an antibiotic resistance cassette and exchange of the plasmid-borne mutant allele with the bacterial chromosome. PCR and recombinational technologies can be exploited to substantially accelerate virtually all steps involved in the gene replacement process. We describe a method for rapid generation of unmarked P. aeruginosa deletion mutants. Three partially overlapping DNA fragments are amplified and then spliced together in vitro by overlap extension PCR. The resulting DNA fragment is cloned in vitro into the Gateway vector pDONR221 and then recombined into the Gateway-compatible gene replacement vector pEX18ApGW. The plasmid-borne deletions are next transferred to the P. aeruginosa chromosome by homologous recombination. Unmarked deletion mutants are finally obtained by Flp-mediated excision of the antibiotic resistance marker. The method was applied to deletion of 25 P. aeruginosa genes encoding transcriptional regulators of the GntR family. While maintaining the key features of traditional gene replacement procedures, for example, suicide delivery vectors, antibiotic resistance selection and sucrose counterselection, the method described here is considerably faster due to streamlining of some of the key steps involved in the process, especially plasmid-borne mutant allele construction and its transfer into the target host. With appropriate modifications, the method should be applicable to other bacteria.
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影响因子：
11.4
作者：
van Aalten, DMF;DiRusso, CC;Wierenga, RK
通讯作者：
Wierenga, RK
DOI：
10.1073/pnas.0337542100
发表时间：
2003-02-18
影响因子：
11.1
作者：
Gust, B;Challis, GL;Chater, KF
通讯作者：
Chater, KF
影响因子：
11.4
作者：
van Aalten, DMF;Dirusso, CC;Knudsen, J
通讯作者：
Knudsen, J
DOI：
10.1073/pnas.120163297
发表时间：
2000-06-06
影响因子：
11.1
作者：
Datsenko, KA;Wanner, BL
通讯作者：
Wanner, BL
影响因子：
11.8
作者：
Wolfgang, MC;Lee, VT;Lory, S
通讯作者：
Lory, S