Molecular cloning of subtilisin gene

Abstract

The purified genomic DNA from Bacillus natto was digested with PstI and eluted by electrophoresis to 2 fractions according to the size. Fraction 1, 2 corresponding 4-20kb, 2-4kb were ligated with a vehicle, pUC9 which was digested with PstI and dephosphorylatedwith CIP. The ligated DNA was transformed into competent Escherichia coli JM83 cells. Around 10,000 colonies were appeared on the selective media containing ampicillin. These transforemdcolonies were tested which hybridized to the probe. The No. FII-2504 colony showed positive spot. The insert size of the plasmid carrying the protease gene was 2.2kb and designated as pSEl. The restriction maps of pSEl was constructed by digesting with Pstl, ScaI, EcoRI, HindIII, respectively. The cell harbouring pSEl showed 1.3-fold high protease activity than control Escherichia coli JM83.