Benson N, Sugiono P, Bass S, Mendelman L V, Youderian P
Genetics. 1986 Sep;114(1):1-14. doi: 10.1093/genetics/114.1.1.
We present a general strategy for the selection of bacterial clones that express DNA-binding activities corresponding to particular DNA recognition sites. The selection uses a "challenge phage" vector, P22 Kn9 arc-amH1605, into which is substituted a synthetic DNA-binding site for a site that controls transcription of the P22 antirepressor (ant) gene. Constitutive synthesis of antirepressor channels a challenge phage into lytic development and efficiently kills an infected host, unless the substituted site is bound by a specific protein; in this case, the challenge phage prefers lysogenic development, and the host survives and acquires an antibiotic-resistance phenotype. Infections with challenge phages carrying the E. coli Lac operator, phage lambda OL1 operator, or synthetic, "idealized" E. coli Trp and Tn10 Tet operators select clones that express each of the corresponding binding activities. The use of challenge phage vectors may be extended to select clones that express eukaryotic DNA-binding activities.
我们提出了一种通用策略，用于选择表达与特定DNA识别位点相对应的DNA结合活性的细菌克隆。该选择使用一种“挑战噬菌体”载体P22 Kn9 arc-amH1605，其中一个合成DNA结合位点取代了控制P22抗阻遏物（ant）基因转录的位点。抗阻遏物的组成型合成会使挑战噬菌体进入裂解发育，并有效地杀死受感染的宿主，除非被取代的位点被特定蛋白质结合；在这种情况下，挑战噬菌体倾向于溶原性发育，宿主存活并获得抗生素抗性表型。用携带大肠杆菌Lac操纵子、噬菌体λ OL1操纵子或合成的“理想化”大肠杆菌Trp和Tn10 Tet操纵子的挑战噬菌体进行感染，可选择表达相应结合活性的克隆。挑战噬菌体载体的应用可能会扩展到选择表达真核DNA结合活性的克隆。
