Construction of a Plasmid Vector for Expression of Bacteriocin N15-encoding Gene and Effect of Engineered Bacteria on Enterococcus Faecalis

Abstract

A 6.09-kb plasmids vector pOri253 was constructed from the plasmid pIL253 (5.2 kb) and a 0.89-kb fragment of oriColE1 from pBluescript II KS. The bifunctional plasmid pOri253 conferred erythromycinresistance in both Escherichia coli and Enterococcus faecalis. It has a unique site for each of EcoRI, BamHI, SalI and PstI derived from pIL253. The plasmid is quite stable in E. faecalis JCM8726 when cultured in Mann Rogosa Sharpe broth without antibiotic. The lactococcal promoter P23 was inserted at one end of the pOri253 multicloning site. Gene expression was assessed by using an entAI gene which coded for bacteriocin N15. The E. faecalis harboring constructed plasmid carrying P23 (pOrient23) had more antibacterial activity than parental E. faecalis JCM8726 or its clone harboring non-P23 containing plasmid (pOrient), as determined by means of an overlay method.

Keywords

• EntAI
• Enterococci
• Shuttle vector
• pIL253