Expression of gene(s) in restricted fragment of lambda DNA in E. coli.

Abstract

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.