In vivo DNA cloning and adjacent gene fusing with a mini-Mu-lac bacteriophage containing a plasmid replicon.

Abstract

A min-Mu phage containing a high copy nubmer plasmid replicon was constructed to clone genes in vivo. A chloramphenicol resistance gene for independent selection and the lacZYA operon to form gene fusions were also incorporated into this pahge. This mini-Mu element can transpose at a high frequency when depressed and it can be complemented by a helper Mu prophage for lytic growth. DNA sequences that are flanked by 2 copies of this min-Mu can be packaged along with them. After infection, homologous recombination can occur between the mini-Mu sequences, resulting in the formation of plasmids carrying the transduced sequences. lac Operon fusions can be formed with promoters and translation initiation sites on the cloned sequences in the resulting plasmids. The utility of this system was demonstrated by cloning genes from 8 different Escherichia coli operons and by identifying lac fusions to the regulated araBAD operon among clones selected for the nearby leu operon.