A 2-COMPONENT T7-SYSTEM FOR THE OVEREXPRESSION OF GENES IN PSEUDOMONAS-AERUGINOSA
A 2-COMPONENT T7-SYSTEM FOR THE OVEREXPRESSION OF GENES IN PSEUDOMONAS-AERUGINOSA
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DOI：
10.1016/0378-1119(92)90600-t
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发表时间：
1992-02-01
期刊：
影响因子：
3.5
通讯作者：
DARZINS, A
中科院分区：
文献类型：
--
作者：
BRUNSCHWIG, E;DARZINS, A
为在非肠道细菌铜绿假单胞菌中高效表达基因，开发了双组分T7表达系统。表达系统的第一个组成部分是一个基于噬菌体的转座因子，它含有一个lacUV 5/lacI（q）调节的T7 RNA聚合酶基因和一个可选择的抗真菌决定簇。该元件，命名为miniD-180，稳定整合到铜绿假单胞菌PAO 1染色体中。该系统的第二个组成部分包括几个改进的广宿主范围表达载体，其含有T7基因10启动子和多克隆位点（MCS）。这些载体（pEB 8、pEB 11和pEB 12）含有T7启动子上游的转录终止子（T1（4））和MCS下游的T7终止子。因为T7启动子在这些载体中有些泄漏，所以构建pEB 14以通过T7 RNA聚合酶的基础水平降低靶基因的转录。该载体含有位于T7启动子转录起始点下游19 bp处的lac操纵子的核心序列，从而提供了双重调节系统。通过将无启动子的氯霉素乙酰转移酶（CAT）盒置于T7启动子的控制下并监测CAT在铜绿假单胞菌的无细胞提取物中的异丙基-β-D-硫代半乳糖苷依赖性积累来证明该系统的实用性。我们观察到诱导后4小时CAT水平增加近60倍，此时该多肽占总可溶性蛋白的20%。
A two-component T7 expression system was developed for efficient expression of genes in the nonenteric bacterium, Pseudomonas aeruginosa. The first component of the expression system is a bacteriophage-based transposable element that contains a lacUV5/lacI(q)-regulated T7 RNA polymerase gene and a selectable antibiotic-resistance determinant. This element, designated miniD-180, was stably integrated into the P. aeruginosa PAO1 chromosome. The second component of this system includes several improved broad-host-range expression vectors containing the T7 gene 10 promoter and multiple cloning site (MCS). These vectors (pEB8, pEB11, and pEB12) contain transcriptional terminators (T1(4)) upstream from the T7 promoter, and T7 terminators downstream from the MCS. Because the T7 promoter is somewhat leaky in these vectors, pEB14 was constructed to decrease transcription of target genes by basal levels of T7 RNA polymerase. This vector contains a core sequence of the lac operator located 19 bp downstream from the transcriptional start point of the T7 promoter, thereby providing a dually regulated system. The utility of this system was demonstrated by placing a promoterless chloramphenicol acetyltransferase (CAT) cassette under control of the T7 promoter and monitoring the isopropyl-beta-D-thiogalactopyranoside-dependent accumulation of CAT in cell-free extracts of P. aeruginosa. We observed up to nearly a 60-fold increase in CAT levels 4 h post-induction, at which time this polypeptide represented up to 20% of the total soluble protein.