IMPROVEMENT OF HOMOLOGOUS RECOMBINATION EFFICIENCY IN E. COLIS.M. AMIN MARASHI , S. YASLIANIFARD , M. ERFANMANESH, P. AFROGH AND E. KALANTAR
Homologous recombination efficiency is dependent to various parameters like recA gene expression, GC content, arabinose and PCR concentration. Based on this we aimed to evaluate the recA and gam genes expression in homologous recombination efficiency in E. coli. In this study, hybrid constructs were prepared with different GC content, arabinose concentration and PCR product by using Wanner Protocol which was electroporated to standard E. coli. Similarly, to evaluate the expression of recA and gam genes, the pKD46 plasmid was transformed to E. coli. The Real-Time PCR for E. coli was performed twice in triplicate and the mean and standard deviations were calculated. Maximum recombinant colonies obtained by 55% GC content. Similarly, the arabinose concentration of 15 mM showed maximum recombinant colonies. The results also showed that as the amount of PCR product for electroporation increases the recombinant colonies resistant to chloramphenicol increased. The results show significant differences in recA & gam gene expression among the standard host and standard host containing pKD46 vector. Quantitative PCR results show that the recA in vector plasmid pKD46 and gam gene expression increased over a hundred times more than the standard host. Using the optimized factors in homologous recombination obtained in this study, can help the researchers in saving time and the expenses in obtaining the best results in the inactivation of genes. So researchers can plan to delete, add or create a mutation on chromosome genes
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