Munich Center for Integrated Protein Science (CIPSM) at the Department of Microbiology, Ludwig-Maximilians-Universität München, 82152 Martinsried, Germany.
J Microbiol Methods. 2012 Dec;91(3):537-43. doi: 10.1016/j.mimet.2012.09.023. Epub 2012 Sep 27.
β-Galactosidase encoded by lacZ remains a popular reporter enzyme. Here, we present three fast and convenient tools that facilitate rapid construction of reporter lacZ fusions. The first enables the simple generation of lacZ (slacZ)-based chromosomally encoded reporter fusions within the lac operon in Escherichia coli using Red®/ET® recombination. The slacZ tool is based on rpsL counter-selection in combination with homologous recombination catalyzed by the λ Red recombinase, and blue/white screening. This permits construction of transcriptional and translational reporter lacZ fusions within a day. The second tool allows the introduction of lacZ reporter fusions into the chromosome by a single-crossover method. The strategy relies on the γ-origin-based suicide vector pNPTS138-R6KT, which can only replicate in λpir E. coli strains. The third tool comprises four pBBR1-based broad-host-range vectors for transcriptional and translational lacZ fusions. The functionality of our toolbox was confirmed by the K(+)-dependent activation of kdp promoter-lacZ fusions in vivo.
β-半乳糖苷酶由 lacZ 编码，仍然是一种流行的报告酶。在这里，我们介绍了三个快速方便的工具，可用于快速构建报告 lacZ 融合物。第一个工具可利用 Red®/ET®重组在大肠杆菌的 lac 操纵子中简单地生成基于 lacZ(slacZ)的染色体编码报告融合物。slacZ 工具基于 rpsL 反向选择，结合 λ Red 重组酶介导的同源重组和蓝/白筛选。这允许在一天内构建转录和翻译报告 lacZ 融合物。第二个工具允许通过单交换方法将 lacZ 报告融合物引入染色体。该策略依赖于基于 γ 起点的自杀载体 pNPTS138-R6KT，它只能在 λpir E. coli 菌株中复制。第三个工具由四个基于 pBBR1 的广谱宿主载体组成，用于转录和翻译 lacZ 融合物。我们的工具包的功能通过体内 K(+)依赖性激活 kdp 启动子-lacZ 融合物得到了证实。
