基于同源双交换的聚球藻PCC 7942基因组大片段无标记删除

UNMARKED LARGE FRAGMENT DELETIONS IN THE GENOME OF SYNECHOCOCCUS ELONGATUS PCC 7942 BASED ON HOMOLOGOUS DOUBLE-CROSSOVER RECOMBINATION

  • 摘要: 在蓝藻合成生物学中, 对大片段进行无标记删除可以加快基因组简化的进程。研究以聚球藻PCC 7942为材料, 基于同源重组技术对基因组中大于10 kb的3个非必需区域进行无标记删除。构建带有两侧同源片段的不可在聚球藻复制的质粒, 利用接合转移将其导入藻细胞获得同源单交换株, 再借助于条件致死基因sacB筛选第二步交换的克隆, 获得无标记删除突变株。研究证明了传统的同源重组和筛选技术可用于蓝藻基因组的大片段无标记删除。

     

    Abstract: In synthetic biology of cyanobacteria, unmarked deletions of large fragments accelerate the process of genome simplification. In this study, three genomic regions of more than 10 kb, namely Synpcc7942_0050-Synpcc7942_0064, Synpcc7942_0233- Synpcc7942_0253 and Synpcc7942_1391-Synpcc7942_1400, were successfully deleted in Synechococcus elongatus PCC 7942 via traditional homologous double crossovers. Non-replicative plasmids with the two flanking homologous fragments were constructed and introduced into cyanobacterial cells to produce single-crossover recombinants. At this step, single crossover recombination occurred in one or both homologous region(s). Next, colonies freed of sacB (a conditional lethal gene in the vector portion) were selected on sucrose plates. Such colonies could be either double-crossover recombinants or revertants, depending on where the second recombination occurred; however, those single crossover recombinants with the plasmid integrated at both homologous regions were inclined to produce mutants rather than revertants. Large fragment deletion mutants were then identified by PCR examinations. This study demonstrated that traditional homologous recombination techniques can be used to generate unmarked large fragment deletions in cyanobacterial genomes.

     

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