Construction and use of SPP1v, a viral cloning vector for Bacillus subtilis

Abstract

A unique BamHI restriction site has been inserted into a nonessential region of the genome of a deletion mutant of phage SPP1. Construction of this phage, designated SPP1 v, required the in vitro conversion of a BclI site to a BamHI site. SPP1 v has been used as a vector phage to clone BamHI, BglII and BclI-generated restriction fragments of DNA. A direct selection for recombinants has been developed. Transfection with SPP1 v requires intact, genomic-length molecules, and cleavage with BamHI destroys the transfecting ability of this DNA. Recombinants in which the BamHI site has been destroyed by ligation to Bg/II or BclI-generated fragments of DNA become resistant to BamHI digestion after ligation and are active in transfection. Cloning of DNA containing BamHI sites has been accomplished by using the enzyme Bst1503I to methylate BamHI sites before insertion, and so to protect them during the BamHI digestion used to select against vector molecules. The in vitro construction of SPP1 v generated XmaIII sites directly adjacent to, and on both sides of the inserted BamHI site. This permits precise excision of cloned DNA even when cloning destroys the BamHI insertion site. Restriction-enzyme generated fragments of DNA in the size range of 0 to 4 Md have been cloned, including a full-length copy of plasmid pUB110, almost the complete sequence of plasmid pBR322, and a sequence of DNA that carried the lambda cos site.