Blue/white screening of recombinant plasmids in Gram-positive bacteria by interruption of alkaline phosphatase gene (phoZ) expression
Blue/white screening of recombinant plasmids in Gram-positive bacteria by interruption of alkaline phosphatase gene (phoZ) expression
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DOI：
10.1016/s0378-1119(98)00396-5
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发表时间：
1998-09-28
期刊：
影响因子：
3.5
通讯作者：
Rubens, CE
中科院分区：
文献类型：
--
作者：
Chaffin, DO;Rubens, CE
筛选重组质粒的细菌转化体的过程通过使用具有视觉可检测的报告基因的载体而变得更加快速和简单。在这样的系统中，当载体携带的指示基因被外源DNA中断时，菌落表型发生改变。虽然lacZ系统已被广泛用于此目的，在E。大肠杆菌，用于革兰氏阳性菌的类似系统仍然不常见。我们已经开发了一种革兰氏阳性克隆载体，利用中断的碱性磷酸酶基因，phoZ，以确定重组质粒。为了促进外源DNA的引入，多克隆位点（MCS）被插入到从衍生phoZ基因（phoZMCS）表达的phoZ碱性磷酸酶的推定信号肽的编码区域的远端，保留与天然蛋白类似的活性。将phoZMCS转移到pJS 3中，pJS 3是一种充分表征的、高拷贝数和宽宿主范围的质粒，以产生pDC 123。在pDC 123中，phoZMCS在组成型表达的tetM和cat启动子的控制下转录连接到氯霉素乙酰转移酶（cat）基因，所述启动子驱动pJS 3中的cat表达。S.无乳链球菌（B群链球菌，GBS）、E.粪链球菌S. pyogenes、S. gordonii和E.含有pDC 123的大肠杆菌在含有5-溴-4-氯-3-吲哚磷酸（X-p）的琼脂上显示蓝色菌落表型，这很容易与含有亲本质粒pJS 3的菌落相区分。将外源DNA导入phoZMCS的MCS中，在E.大肠杆菌和GBS在含有X-p的琼脂上，并允许区分含有重组质粒的转化体与那些保持自身退火或未切割载体的转化体。我们已经使用pDC 123从质粒pCER 111亚克隆cpsE基因，该质粒携带GBS荚膜多糖合成位点的9.0 kb片段。通过直接电穿孔到GBS菌株A909中来分离含有cpsE的质粒pDC 123，通过“蓝/白色”筛选来选择含有重组质粒的转化体，而不使用中间宿主。这种新的克隆载体将提高在革兰氏阳性菌中进行重组DNA实验的效率。(C)1998 Elsevier Science B. V.保留所有权利。
The process of screening bacterial transformants for recombinant plasmids is made more rapid and simple by the use of vectors with visually detectable reporter genes. In such systems, an alteration in colony phenotype occurs when a vector-borne indicator gene is interrupted with exogenous DNA. Although the lacZ system has been used extensively for this purpose in E. coli, analogous systems for use in Gram-positive bacteria remain uncommon. We have developed a Gram-positive cloning vector that utilizes the interruption of an alkaline phosphatase gene, phoZ, to identify recombinant plasmids. To facilitate introduction of foreign DNA, a multiple cloning site (MCS) was inserted distal to the region coding for the putative signal peptide of phoZ Alkaline phosphatase expressed from the derivative phoZ gene (phoZMCS) retained activity similar to that of the native protein. The phoZMCS was transferred to pJS3, a well-characterized, high-copy number, and broad-host-range plasmid, to produce pDC123. In pDC123, phoZMCS was transcriptionally linked to the chloramphenicol acetyl transferase (cat) gene under the control of the constitutively expressed tetM and cat promoters that drive cat expression in pJS3. S. agalactiae (Group B streptococci, GBS), E. faecalis, S. pyogenes, S. gordonii, and E. coli containing pDC123 displayed a blue colonial phenotype on agar containing 5-bromo-4-chloro-3-indolyl phosphate (X-p), which was readily distinguished from that of colonies containing the parent plasmid pJS3. Introduction of foreign DNA into the MCS of phoZMCS produced a white colonial phenotype in E. coli and GBS on agar containing X-p and allowed discrimination between transformants containing recombinant plasmids versus those maintaining self-annealed or uncut vector. We have used pDC123 to subclone the cpsE gene from the plasmid pCER111, which carries a 9.0-kb fragment of the GBS capsular polysaccharide synthesis locus. The plasmid pDC123 containing cpsE was isolated by direct electroporation into GBS strain A909 with selection of transformants containing recombinant plasmids achieved by 'blue/white' screening, without the use of an intermediate host. This new cloning vector should improve the efficiency of performing recombinant DNA experiments in Gram-positive bacteria. (C) 1998 Elsevier Science B.V. All rights reserved.