Abstract:
Due to its robustness to environmental stresses and fast growth, Synechococcus elongatus UTEX2973 is developed as a new model for researches on cyanobacterial molecular biology and biotechnology. However, systematic genetic modifications of S. elongatus UTEX2973 were hindered by the lack of effective genetic manipulation tools, especially available counter-selection markers. Here, six synthetic counter-selection markers (SCOMs) were assembled by fusing six toxin genes from either Escherichia coli or cyanobacteria with a theophylline-inducible promoter. The SCOMs containing SYNPCC7002_G0085 from Synechococcus sp. PCC7002 or mazF from E. coli were proved to be inducible by theophylline in S. elongatus UTEX2973. By using the mazF-based SCOM, the neutral locus 1 and 23 small regulatory RNAs were completely deleted from the genome of S. elongatus UTEX2973 after one round of selection with both kanamycin and theophylline. The genetic tools developed in this work will facilitate future researches on molecular genetics and synthetic biology in S. elongatus UTEX2973. KEY POINTS: • Two inducible counter-selection markers are lethal to S. elongatus UTEX2973. • The counter-selection marker benefits the gene targeting in S. elongatus UTEX2973. • Twentry-three small regulatory RNAs were fully deleted via the novel gene targeting method.
摘要:
由于其对环境压力和快速增长的鲁棒性,长毛球藻UTEX2973是蓝藻分子生物学和生物技术研究的新模型。然而,由于缺乏有效的遗传操作工具,阻碍了长尾沙门氏菌UTEX2973的系统遗传修饰,特别是可用的反选择标记。这里,通过将来自大肠杆菌或蓝细菌的六个毒素基因与茶碱诱导型启动子融合,组装了六个合成反选择标记(SCOM)。含有来自Synechococcussp。的SYNPCC7002_G0085的SCOM。来自大肠杆菌的PCC7002或mazF被证明可被茶碱诱导。通过使用基于mazF的SCOM,在用卡那霉素和茶碱进行一轮选择后,中性基因座1和23个小调节RNA从长龙链球菌UTEX2973的基因组中完全缺失.在这项工作中开发的遗传工具将促进S.elongatusUTEX2973分子遗传学和合成生物学的未来研究。关键点:•两个诱导型反选择标记对长尾链球菌UTEX2973是致死的。•反选择标记有利于长毛链球菌UTEX2973中的基因靶向。•通过新的基因打靶方法完全删除了Twentry-3个小调节RNA。