Genetic transformation and vector development in zymomonas mobilis ATCC 10988 by plasmid $RP_4$ DNA

Abstract

Plasmids of $RP_4$ and pBR322 in strains of $\underline{Escherichia} $\underline{coli}$ were isolated and tried to transform into cells of $\underline{Zymomonas mobilis}$ for the purpose of developing a vector which may be used later for cloning genes in the host organism. $RP_4$ and pBR322 plasmid DNA were isolated from $\underline{E}$. $\underline{coli}$ C600 strains, respectively, and the transformation was carried out by incubating $RP_4$ or pBR322 with $\underline{Zymomonas}$ cells in $MxCa^{++}$ buffer for 60 min under iced temperature. After the incubation, the host cells were plated out in selective media and incubated for 6 days at 30``C to allow development of colonies. The occurrence of transformants were confirmed. Conditions of transformation were tried to change and their effect on transformation frequency was also observed to find optimal conditions for transformation. It was found that the plasmid $RP_4$ of $\underline{E}$. $\underline{coli}$ origin could transfer freely to $\underline{Z}$. $\underline{mobilis}$ cells, whereas, pBR322 could not. The optimum conditions for the transformation of $RP_4$ into $\underline{Z}$. $\underline{mobilis}$ cells were; (1) at the late exponential phase of the host cells, (2) at 50 mM $CaCl_2$ concentration in $MxCa^{++}$ buffer, (3) with heat shock of 30``C for 2 min, (4) expression time of 60 min, and (5) DNA concentration of 3.5 ug per ml. By the comparison minimum inhibitory concentration values of $\underline{Z}$. $\underline{mobilis}$ and $\underline{E}$. $\underline{coli}$ ($RP_4$) against Kanamycin and tetracycline after transformation of $RP_4$ plasmid, it was concluded that the expression activity of $RP_4$ in $\underline{Z}$. $\underline{mobilis}$ was reduced to half than in $\underline{E}$. $\underline{coli}$. The $RP_4$ plasmid was also observed to maintain stably in $\underline{Z}$. $\underline{mobilis}$ cells during one-batch growth, minimally. Therefore, the plasmid $RP_4$ obtained fr...