Expression Analyses of Methylase Genes of Ppamc125 In Methylating Pbbre194 Prot-CM1 Plasmid In Escherichia Coli DH5a and TOP10

Abstract

Abstract In molecular biological techniques, bacterial transformation is an essential process to strain improvement. However, transformation rate into wild type bacteria is commonly low because of the cell defense mechanism of the bacteria. Restriction modification (RM) in bacteria cells can prevent the introduction of recombinant plasmids into target bacteria. Previously, the transformation of recombinant shuttle vector pBBRE194 containing protease gene into wild type Bacillus halodurans CM1 (pBBRE194 prot-CM1) has been conducted. However, the transformation rate seemed low, and the stable recombinant clones could not be obtained. Therefore, in vivo methylation of this plasmid in E. coli has to be done before genetic transformation into the wild type bacterium, to obtain stable recombinant CM1 clones. In this study, a plasmid with artificial modification (pPAMC125) harboring genes encoding for the modification enzymes (methylases) from another strain, B. halodurans C-125, and a shuttle E.coli-Bacillus vector pBBRE194 prot-CM1 plasmid were transformed simultaneously into E. coli DH5a and TOP10, respectively. The expression of methylation enzymes genes carried by pPAMC125 in the presence of different L-arabinose concentration was observed via SDS-PAGE analyses. The analysis of the effect of these genes expression towards plasmid pBBRE194 prot-CM1 was performed by digesting the plasmid with the extracted cell of B. halodurans CM1. The digestion pattern was analyzed via DNA electrophoresis. As a result, compared to E. coli DH5a, E. coli TOP10 exhibited better condition for methylase genes expression with the result that pBBRE194 prot-CM1 plasmid was not be completely degraded by B. halodurans CM1 restriction endonuclease.