CONSTRUCTION OF A SHUTTLE VECTOR BETWEEN E.COLI ANDB. SUBTILIS
A stable shuttle vector was constructed by ligating plasmid pNO1523 with plasmid pUB110. The new vector, pNU2, approximately 9.4 kb was capable of replicating in both E.coli and B. subtilis. The shuttle vector confers ampicillin and kanamycin resistance for E.coli and kanamycin resistance for B. subtilis. Transformation frequency was 4×102 transformants per microgram of DNA in competent B. subtilis.Restriction enzyme analyses of the vectors isolated from both microorganisms showed that they were identical. Also, the shuttle vector is stable for insertional inactivation and carries many unique restriction sites located outside the antibiotic resistance genes.