Schaaper R M, Danforth B N, Glickman B W
Gene. 1985;39(2-3):181-9. doi: 10.1016/0378-1119(85)90312-9.
We have developed a procedure to efficiently recover lac repressor mutations (lacI-) from F'lac onto a single-stranded M13 phage vector. The recovery is based on homologous recombination between F'lac and an M13lac vector. This vector, mRS81, carries the entire Escherichia coli lacI gene as well as the adjacent alpha-complementation region of the lacZ gene, inserted in the AvaI site of the M13 ori region. It also carries a single point mutation in lacZ- alpha which abolishes its alpha-complementing ability. Recovery of lacI- genes from F is based on the conversion of this lacI+Z- alpha phage to lacI-Z+ alpha by recombination with F'lacI-Z+. This double exchange restores its alpha-complementing ability in the absence of any inducer of the lac operon. Detection requires a lacI- alpha-complementation host, which was also constructed in this study. The procedure was developed to obtain rapid nucleotide sequence information on large collections of lacI mutants for the purpose of studying mutational mechanisms and specificities.
我们已经开发出一种程序，可有效地将F'lac上的乳糖阻遏物突变（lacI-）恢复到单链M13噬菌体载体上。这种恢复是基于F'lac与M13lac载体之间的同源重组。该载体mRS81携带整个大肠杆菌lacI基因以及lacZ基因的相邻α-互补区域，插入到M13 ori区域的AvaI位点。它在lacZ-α中还携带一个单点突变，使其失去α-互补能力。从F中恢复lacI-基因是基于这种lacI+Z-α噬菌体通过与F'lacI-Z+重组而转化为lacI-Z+α。这种双交换在没有任何乳糖操纵子诱导剂的情况下恢复其α-互补能力。检测需要一个lacI-α-互补宿主，本研究中也构建了该宿主。开发该程序的目的是为了研究突变机制和特异性，从而快速获得大量lacI突变体的核苷酸序列信息。
