Kuang D R, He J K, Ju Q D, Gong Q H, Wang M Z, Zhu X L
Sci Sin. 1981 Feb;24(2):256-63.
Restriction endonucleases EcoR1 and BamH1 are used to produce fragments pBR322C (375 bp) and pBR322B (3987 bp) from pBR322, and to produce lambda F2A (65.6--71.3% of lambda DNA, 2679 bp) and lambda F2B (71.3--81% of lambda DNA, 4559 bp) from EcoR1 restriction fragment lambda F2 (65.6--81% of lambda DNA) of lambda cI857S7 DNA. By recombining pBR322B and lambda F2B in vitro, a new plasmid called pCB2 carrying promoters and structural genes cI and cro is constructed. The desired strain with pCB2 is selected from 338 transformants for its AprTcs and for its immunity to lambda infection. The length of the pCB2 DNA molecule is 2.66 +/- 0.33 micrometers and its MW is 5.51 +/- 0.68 x 10(6)d, as determined by electron microscope and agarose gel electrophoresis. The lengths of single strands and the double strands of the heteroduplex formed between lambda F2 and linear pCB2 (EcoR1 digested) agree well with the original design for its construction, From the above data, we come to the conclusion that pCB2 we constructed is a new plasmid with cI and/or cro gene expressed in E. coli.
限制性内切酶EcoR1和BamH1用于从pBR322产生片段pBR322C（375碱基对）和pBR322B（3987碱基对），并从λcI857S7 DNA的EcoR1限制性片段λF2（λDNA的65.6 - 81%）产生λF2A（λDNA的65.6 - 71.3%，2679碱基对）和λF2B（λDNA的71.3 - 81%，4559碱基对）。通过在体外将pBR322B和λF2B重组，构建了一种名为pCB2的新质粒，其携带启动子以及结构基因cI和cro。从338个转化体中筛选出具有pCB2的所需菌株，依据其氨苄青霉素抗性和四环素敏感性以及对λ感染的免疫性。通过电子显微镜和琼脂糖凝胶电泳测定，pCB2 DNA分子的长度为2.66±0.33微米，其分子量为5.51±0.68×10⁶道尔顿。λF2与线性pCB2（经EcoR1消化）形成的异源双链的单链和双链长度与构建它的原始设计吻合良好。根据上述数据，我们得出结论，我们构建的pCB2是一种在大肠杆菌中表达cI和/或cro基因的新质粒。
