EFFECT OF SIZE, (\TEXTIT{ORI}) TYPE, AND DNA CER}) FRAGMENT ON STABILITY OF PLASMID CARRYING SOD GENE

• Main Author: Kesuma, Suryanata
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/78942
• Institution: Institut Teknologi Bandung

Problems that frequently occur in the production of proteins recombinant are decreasing segregation and structural stability of plasmid. Those will decrease protein production. Stability of plasmid segregation and structure are affected by structure, number of copies and size of plasmid. This study was aimed to evaluate the effect of size, ori type, and DNA cer fragment on plasmid segregation and structural stability, sod gene expression, and rSOD activity. Among plasmids that encoding rSOD are pJexpress_sod, pBM_sod, pCAD sod, and pCAD sod dapD cer. Stability of plasmid segregation was analyzed using replica platting, stability of plasmid structural used by agarose gel electrOphoresis, sod gen expression used by SDS-PAGE and rSOD activity use&by zymography with natifPAGE. The stability of the pCAD sod dapD_cer segregation was the best and pJexpress sod was the worst among pBM sod and pCAD sod, the decrement of plasmid stability become less than 90% occur in generation time of 20 to 30 and 75 to 85. DNA cer fragment were inserted on pCAD sod dapD cer was able to improve the stability of segregation at the point of less than 90% in generation of 85. Plasmid size does not change when the plasmid segregation become less 90%. rSOD protein size when segregation stability less than 90% of each plasmid was stable, that is 23.4 kDa. The intensity of the rSOD protein bands from plasmid pCAD sod dapD cer is most small and pBM sod is greatest when the stability of segregation less than 90%. In addition, it has been confirmed that the enzymatic activity of rSOD decreased when the stability of plasmid segregation is less than 90% and and rSOD from plasmid pCAD sod is the most decreased activity that is 50%. The results show those size, ori type, and DNA cer fragment affect the stability of segregation, the intensity of the protein resulted, and rSOD activity, but not for the stability of the size on the pJexpress_sod, pBM sod, pCAD sod dan pCAD sod dapD_cer plasmids.