Isolation of Bacillus subtilis genes from a charon 4A library

Abstract

A library of Bacillus subtilis chromosomal deoxyribonucleic acid (DNA) was constructed, using lambda charon 4A as a cloning vector. Partially cleaved Bacillus subtilis DNA was prepared by partial methylation with EcoRI methylase, followed by complete EcoRI endonuclease digestion. More than 95% of the phage particles carried B. subtilis DNA inserts. When this library was screened for transforming activity, using competent cells, 70% of the genetic markers tested were found in a sample of 1,710 plaques. Cloned genetic loci were found to be about 100-fold more efficient in transforming activity than chromosomal DNA. Intact phage particles containing the pheA locus were found to be able to transform competent recipients with approximately the same efficiency as phage DNA. Transformation by intact particles was insensitive to deoxyribonuclease.