Integration-proficient plasmids for Pseudomonas aeruginosa:: Site-specific integration and use for engineering of reporter and expression strains
Integration-proficient plasmids for Pseudomonas aeruginosa:: Site-specific integration and use for engineering of reporter and expression strains
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DOI：
10.1006/plas.1999.1441
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发表时间：
2000-01-01
期刊：
影响因子：
2.6
通讯作者：
Schweizer, HP
中科院分区：
文献类型：
--
作者：
Hoang, TT;Kutchma, AJ;Schweizer, HP
建立了一种将外源DNA片段整合到铜绿假单胞菌基因组特定位点的改良方法。该方法依赖于两个整合熟练的载体，mini-CTX 1和mini-CTX 2。这两种载体含有（1）四环素（泰特）选择标记，（2）用于接合介导质粒转移的oriT，（3）pMB 1衍生的复制起点，（4）修饰的phi CTX整合酶（int）基因，（5）侧翼为T4转录终止序列（Ω元件）的通用多克隆位点（MCS），以及（6）phi CTX附着位点。MCS和Ω元件的侧翼是酵母Flp重组酶靶位点，其允许通过Flp重组酶从整合体的基因组中体内切除不需要的质粒骨架序列，包括泰特和int。在mini-CTX 2载体中，int转录由强trc启动子驱动，该启动子由也包含在质粒上的lacl（q）编码的Lac阻遏物调节。在接合转移时，mini-CTX 1和mini-CTX 2分别以10（-8）和10（-7）的频率整合。通过将自诱导物（AI）调节的lasB-lacZ和rhlA-lacZ融合体插入到野生型和AI合酶突变体中，证明了整合载体用于基因融合分析的有用性。在野生型中，融合体以细胞密度依赖性方式响应，并且在AI合酶突变体中，两种融合体的表达都大大减少或消除。最后，构建含有Lac阻遏物控制下的T7聚合酶基因的表达盒，将其整合到铜绿假单胞菌染色体中，并用于表达六组氨酸标记的铜绿假单胞菌AI合酶RhII。(C)北京大学出版社.
An improved method for integration of exogenous DNA fragments at a defined site within the genome of Pseudomonas aeruginosa was developed. The method relies on two integration-proficient vectors, mini-CTX1 and mini-CTX2. These two vectors contain (1) a tetracycline (tet) selectable marker, (2) an oriT for conjugation-mediated plasmid transfer, (3) the pMB1-derived origin of replication, (4) a modified phi CTX integrase (int) gene, (5) a versatile multiple cloning site (MCS) flanked by T4 transcriptional termination sequences (Omega elements), and (6) the phi CTX attachment site. The MCS and Omega elements are flanked by yeast Flp recombinase target sites that allow in vivo excision of unwanted plasmid backbone sequences, including tet and int, from the genome of integrants by Flp recombinase. In the mini-CTX2 vector int transcription is driven from the strong trc promoter, which is regulated by the Lac repressor that is encoded by lacl(q) also contained on the plasmid. Upon conjugal transfer,mini-CTX1 and mini-CTX2 integrated at frequencies of 10(-8) and 10(-7), respectively. The usefulness of the integration vectors for gene fusion analyses was demonstrated by chromosomal insertion of autoinducer (AI)-regulated lasB-lacZ and rhlA-lacZ fusions into wild-type and AI synthase mutants. In wild-type, the fusions responded in a cell density-dependent manner and expression of both fusions was either greatly reduced or abolished in AI synthase mutants. Finally, an expression cassette containing the T7 polymerase gene under Lac repressor control was constructed, integrated into the P. aeruginosa chromosome, and used to express the hexahistidine-tagged P. aeruginosa AI synthase RhlI. (C) 2000 Academic Press.