Rapid and Liquid-Based Selection of Genetic Switches Using Nucleoside Kinase Fused with Aminoglycoside Phosphotransferase

The evolutionary design of genetic switches and circuits requires iterative rounds of positive (ON-) and negative (OFF-) selection. We previously reported a rapid OFF selection system based on the kinase activity of herpes simplex virus thymidine kinase (hsvTK) on the artificial mutator nucleoside dP. By fusing hsvTK with the kanamycin resistance marker aminoglycoside-(3’)-phosphotransferase (APH), we established a novel selector system for genetic switches. Due to the bactericidal nature of kanamycin and nucleoside-based lethal mutagenesis, both positive and negative selection could be completed within several hours. Using this new selector system, we isolated a series of homoserine lactone-inducible genetic switches with different expression efficiencies from libraries of the Vibrio fischeri lux promoter in two days, using only liquid handling.

基因开关与基因回路的进化设计需要多轮正负（开/关）筛选。此前我们报道了一套基于单纯疱疹病毒胸苷激酶（herpes simplex virus thymidine kinase, hsvTK）对人工诱变核苷dP的激酶活性的快速负筛选系统。通过将hsvTK与卡那霉素抗性标记氨基糖苷-(3’)-磷酸转移酶（aminoglycoside-(3’)-phosphotransferase, APH）融合，我们构建了一套用于基因开关研究的新型筛选系统。鉴于卡那霉素的杀菌特性与核苷类致死诱变作用，正负筛选均可在数小时内完成。利用这套新型筛选系统，我们仅通过液体操作，在两天内从费氏弧菌lux启动子文库中分离得到一系列具有不同表达效率的高丝氨酸内酯诱导型基因开关。