A new system to place single copies of genes, sites and lacZ fusions on the Escherichia coli chromosome

To place a single-copy lacZ fusion on the E. coli chromosome, a method was developed based on in vivo homologous DNA recombination through P1 transduc tion. The fusions, initially constructed on plasmids, are crossed to lambda lacZ fusion vectors which are then lysogenized at the chromosomal lambda a tt site. The features of the new system are: (1) lambda lysogens carrying t he fusion are made without regard for copy number; (2) P1 transduction from the lysogenic strain into an appropriate recipient generates the single-co py fusion; (3) The lacZ fusion has no prophage associated with it; (4) the lacZ fusion can be transferred by P1 transduction to other strains, simply by selecting for an antibiotic marker; (5) the system can be widely applied to construct single copies of any gene or site placed between bla and lacZ on the standard lacZ fusion plasmid vectors; and (6) the single-copy const ruct flanked by prophage atl sites can be excised by site-specific recombin ation to generate non-replicating circular DNA of the clone or a cell cured of the construct. (C) 1998 Elsevier Science B.V. All rights reserved.