Scarless and sequential gene modification in Pseudomonas using PCR product flanked by short homology regions.
Scarless and sequential gene modification in Pseudomonas using PCR product flanked by short homology regions.
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使用侧接短同源区域的 PCR 产物对假单胞菌进行无痕连续基因修饰
DOI：
10.1186/1471-2180-10-209
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发表时间：
2010-08-03
期刊：
影响因子：
4.2
通讯作者：
Liu, Jianhua
中科院分区：
文献类型：
--
作者：
Liang, Rubing;Liu, Jianhua
背景：λ Red重组系统已被用于在各种细菌和真菌中扩增染色体基因。该程序包括电穿孔的聚合酶链反应（PCR）片段含有抗生素盒两侧的同源区域的目标基因座到一个菌株，可以表达的lambda Red蛋白（Gam，Bet，Exo）。结果：在这里，一个无瘢痕的基因修饰策略的基础上的红色重组系统已开发到修改假单胞菌基因组DNA通过顺序删除多个目标。这一过程由质粒pRKaraRed介导，质粒pRKaraRed编码由PBAD启动子调控的Red蛋白，PBAD启动子在铜绿假单胞菌和其他细菌中具有功能。首先用目的基因替换sacB-bla盒两侧的短同源区（50 bp），然后用PCR片段替换该标记基因盒，得到目标基因缺失的基因组，没有任何残留，周围区域也没有变化。对参与吩嗪衍生物绿脓菌素合成和调控途径的20个基因进行了修饰，包括一个单点突变和两个大操纵子的缺失。重组效率为88%~ 98%。多基因修饰也实现了，产生的三基因缺失菌株PCA（PAO 1，DeltaphzHDeltaphzMDeltaphzS），它可以产生另一个吩嗪衍生物，吩嗪-1-羧酸（PCA），efficiently and exclusively.Conclusions：这种基于λ Red的技术可以用来产生无瘢痕和顺序的基因修饰突变体的铜绿假单胞菌有效地，使用一步PCR产物侧翼短的同源区域。单点突变，无疤痕的基因删除可以在不到三天的时间内轻松实现。该方法为高效构建铜绿假单胞菌基因工程菌提供了一条新途径，也为进一步研究铜绿假单胞菌的调控网络提供了新的思路。
Background:The lambda Red recombination system has been used to inactivate chromosomal genes in various bacteria and fungi. The procedure consists of electroporating a polymerase chain reaction (PCR) fragment containing antibiotic cassette flanked by homology regions to the target locus into a strain that can express the lambda Red proteins (Gam, Bet, Exo).Results:Here a scarless gene modification strategy based on the Red recombination system has been developed to modify Pseudomonas genome DNA via sequential deletion of multiple targets. This process was mediated by plasmid pRKaraRed encoding the Red proteins regulated by PBAD promoter, which was functional in P. aeruginosa as well as in other bacteria. First the target gene was substituted for the sacB-bla cassette flanked by short homology regions (50 bp), and then this marker gene cassette could be replaced by the PCR fragment flanking itself, generating target-deleted genome without any remnants and no change happened to the surrounding region. Twenty genes involved in the synthesis and regulation pathways of the phenazine derivate, pyocyanin, were modified, including one single-point mutation and deletion of two large operons. The recombination efficiencies ranged from 88% to 98%. Multiple-gene modification was also achieved, generating a triple-gene deletion strain PCA (PAO1, DeltaphzHDeltaphzMDeltaphzS), which could produce another phenazine derivate, phenazine-1-carboxylic acid (PCA), efficiently and exclusively.Conclusions:This lambda Red-based technique can be used to generate scarless and sequential gene modification mutants of P. aeruginosa efficiently, using one-step PCR product flanked by short homology regions. Single-point mutation, scarless deletion of genes can be achieved easily in less than three days. This method may give a new way to construct genetically modified P. aeruginosa strains more efficiently and advance the regulatory network study of this organism.
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影响因子：
--
作者：
Murphy KC;Campellone KG
通讯作者：
Campellone KG
DOI：
10.1073/pnas.121164898
发表时间：
2001-06-05
影响因子：
11.1
作者：
Ellis, HM;Yu, DG;Court, DL
通讯作者：
Court, DL
影响因子：
3.1
作者：
Beloin, C;Deighan, P;Dorman, CJ
通讯作者：
Dorman, CJ
DOI：
10.1152/ajplung.00086.2003
发表时间：
2003-09-01
影响因子：
4.9
作者：
Denning, GM;Iyer, SS;Britigan, BE
通讯作者：
Britigan, BE
影响因子：
3.2
作者：
ESSAR, DW;EBERLY, L;CRAWFORD, IP
通讯作者：
CRAWFORD, IP