This study introduces mCloverBlaster as a genetic tool to create deletions and transcriptional and translational fusions in bacterial genomes using recombineering. The major advantage of this system is that it can be used to make deletions and fusions without leaving a selectable marker on the chromosome. mCloverBlaster has a kanamycin resistance cassette with an I-SceI restriction site flanked by fragments of the gene for the mClover3 fluorescent protein including direct repeats of mClover3 sequence on both sides of the kanamycin resistance gene. The mCloverBlaster sequence is introduced into the chromosome using lambda red recombineering, expression of I-SceI creates a double stranded break in the kanamycin resistance cassette that initiates a recombination event that can occur in the mClover3 repeats. This recombination results in the simultaneous removal of the kanamycin resistance gene and the restoration of a functional mClover3 gene that can be used as a reporter. Here, this system was used to replace the rcsB stress response gene in Serratia marcescens. The resulting strain was tested for mClover3 fluorescence as a reporter for rcsB gene expression and evaluated for pigment biosynthesis. In summary, mCloverBlaster is a molecular genetic tool to make markerless mClover3 fusions and gene deletions.

本研究将mClover Blaster 作为一种遗传工具，使用重组工程在细菌基因组中创建缺失以及转录和翻译融合。该系统的主要优点是它可以用于进行缺失和融合，而不会在染色体上留下可选择的标记。mClover Blaster 具有卡那霉素抗性盒，其 I- Sce I 限制性位点两侧是 mClover3 荧光蛋白基因片段，包括卡那霉素抗性基因两侧的mClover 3 序列的直接重复。使用 lambda red 重组工程将mClover Blaster 序列引入染色体，表达 I- Sce我在卡那霉素抗性盒中创建了一个双链断裂，启动了mClover3重复序列中可能发生的重组事件。这种重组导致卡那霉素抗性基因的同时去除和可用作报告基因的功能性mClover3基因的恢复。在这里，该系统用于替换粘质沙雷氏菌中的rcsB应激反应基因。测试所得菌株的 mClover3 荧光作为rcsB基因表达的报告基因，并评估色素生物合成。总之，mClover Blaster 是一种分子遗传工具，可以进行无标记的mClover3融合和基因缺失。