Construction of an expression shuttle vector for Escherichia coli and Bacillus subtilis

Subject Areas : Bacteriology

Mohammad Reza Rashidi ¹ , Majid Moghbeli ²

1 - کارشناس ارشد، دانشگاه آزاد اسلامی، واحد دامغان، دانشکده علوم زیستی، گروه میکروبیولوژی
2 - استادیار، دانشگاه آزاد اسلامی، واحد دامغان، دانشکده علوم زیستی، گروه میکروبیولوژی

Received: 2013-03-30 Accepted : 2013-05-31 Published : 2013-10-01

Keywords: Escherichia coli, Cloning, Bacillus subtilis, Shuttle vector,

Abstract :

Background and Objectives: Bacillus subtilis is considered as a suitable host for gene cloning and protein expression due to non-pathogenicity and ability to secrete high amounts of proteins. However, transformation efficiency of ligated plasmid DNA into competent cells of B. subtilis is low, as compared to that into CaCl2 shocked cells of Escherichia coli. Therefore, cloning the target in E. coli using a shuttle vector before transfer into B. subtilis by a high amount of hybrid vector is more efficient. The aim of this study is construction a shuttle vector using pW980 and pET-16b plasmids for gene cloning and expression in B. subtilis. Materials and Methods: pWB980 plasmid was isolated from its host using alkaline lysis method. It was undergone a double digestion to obtain the segment of interest using EcoRI  and SnaBI enzymes. Then a special fragment of pET-16b plasmid was also amplified by PCR and was double digested. Ligation reaction was performed between these two segments and then it was transferred to E. coli TOP10. Following screening the cells containing shuttle vector, plasmid was extracted and was transferred to B. subtilis WB600 by an effective method. Results: The resulting shuttle vector replicated easily in both hosts. It showed good stability in B. subtilis, which is helpful for its maintenance in its host. Conclusion: In this study, the resulting shuttle vector - pMR12 - had 8 unique cloning site in its polylinker and a suitable size (5.4 kb), which make it possible to simply gene cloning into it. This is the first report of construction of an expression shuttle vector for Escherichia coli and Bacillus subtilis in Iran.