Cloning and expression of the ilvB gene of Escherichia coli K-12

Abstract

A plasmid containing the ilvB operon, which codes for acetohydroxy acid synthase I of Escherichia coli K-12, was isolated using a ligated mixture of DNA from plasmid pBR322 and FilvB4 treated with endonuclease SalI. A shortened derivative of this plasmid was isolated by cloning a 3.4 kb bacterial fragment into plasmid pKEN005 to yield plasmid pTCN12. The orientation of the ilvB operon relative to plasmid genes was determined by restriction enzyme mapping. Measurement of the level of the product of the ilvB gene, acetohydroxy acid synthase I, indicated that plasmid pTCN12 contained a functional ilvB promoter and control region. The DNA frm this plasmid was used as a probe to show that the rate of synthesis of ilvB mRNA was proportional to the levels of acetohydroxy acid synthase I.