Construction Of Prokaryote Transformation System And Gene Cione Of Bacillus Subtilis α-Amylase

By the simple .modified CaCl2 method, all factor that affected thetransformation efficiency were studied. Based on these research results, the high efficiency competent cells were prepared and α -amylase gene of Bacillus subtilis was cloned by the shotgun method..1 Many effect factors of the transformation efficiency were studied through the modified CaCl2 method .Firstly , the factors in the course of preparation competent cells were studied .The medium , the pH valueof the medium , cell density ,Mg2+ were important effect factors. Based on these research , the high efficiency competent cells (transformation efficiency was 10'' transformants per μg ks plasmids DNA ) were made. Then , the factors in the transformating process were studied . The transformation efficiency kept constant as the DNA concentration increased from lpg/μL to 1ng/μl. As the DNA concentration increased from 1ng/μl to 5001ng/μl, the transformation efficiency decreased . The transformants were most at the lOng/μl DNA concentration .and it was showed the lOng/μl was the saturation concentration .Bigger than the 7.3kb , the plasmid size began limiting the transformtion efficiency . Other factors such as the heat pulse temperature , heat pulse time , ventilating conditions , transformation buffer , ice bath time were minor factors .But the heat pulse was necessary when the high efficiency was demanded .2 The transformation system was constructed as the three methods to prepare the competent cells (Inoue method , modified CaCl2 method .colony method), three transformation style (heat pulse method , instant transformation , 37 ℃ water bath transformtion ) and different transformation experiment combined :The system was simple ..concrete and was applied to the gene clone of the Bacillus subtilis α-amylase.3 The a -amylase gene of Bacillus subtilis was cloned by shotgun method .After the genome DNA of Bacillus subtilis were digested for 30 minutes with 0.3U Sau3AI enzyme ,the 3-7kb targets DNAs were extracted .The targets DNAs, the ksplasmid vector which were digested with BamHI enzyme and dephosphorylated with CIP were mixed. Then T4DNA ligase buffer ,pure water were added to 20 ul reaction volume . After 16 to 18 h at 14 @ to 16@, the reaction volume was enlarged to 50 μl, again for 16-18h, then transformation with high efficiency competent cells , the LBSP used as identifying medium, the a -amylase gene was cloned and identified through plasmid detection , plasmid transformation .physical map analysesed.