ANNOUNCEMENTS
Gene cloning and expression in Escherichia coli (E. coli) are fundamental techniques in biotechnology and molecular biology. This thesis explores the cloning of gene X using restriction digestion-ligation technique and its subsequent expression in E. coli. The cloning process involves several key steps, including the isolation of the vector, amplification of the gene X, restriction enzyme digestion of both the vector and the gene X, ligation of the insert into the vector, making the cells competent and its transformation, and selection of positive clones via plating on media containing antibiotic. For further validation of successful recombination and transformation, Colony PCR and restriction digestion was also done. E. coli was then induced with IPTG to express protein. Subsequently, the expression of gene X in E. coli was analysed via SDS-PAGE. We also looked for optimization of expression conditions and characterization of the recombinant protein. This study demonstrates the successful cloning and expression of gene X, highlighting its potential applications in biotechnological and biomedical research. The work has been done to explore the optimizing conditions for a 5 L bio fermenter. Current work also deep dives onto selection of our host organism, particular strain and vector preparation and clone selection for protein production for industrial application.
Keywords: Escherichia coli, cloning, ligation, transformation, induction, recombinant.
