PENGARUH PLASMID pCAD_sod DAN TURUNANNYA TERHADAP PROFIL PERTUMBUHAN Escherichia coli TOP10, EKSPRESI GEN sod DAN STABILITAS PLASMID

• Main Author: Santi, Citta
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/44271
• Institution: Institut Teknologi Bandung

In order to produce recombinant proteins, an expression vector which carries expression cassette and DNA elements to maintain vector stability and selection system is required. pCAD_sod plasmid is one vector constructed in the Laboratory of Pharmaceutical Biotechnology, School of Pharmacy ITB to produce recombinant hybrid manganese superoxide dismutase (SOD) from Staphylococcus equorum (rMnSODSeq) in Escherichia coli. The plasmid utilizes ampicillin antibiotic selection system and dps autoinduction which is active in stationary phase. Two derivates of pCAD_sod plasmid were constructed to carry cer DNA fragment to improve plasmid stability (pCAD_sod::cer) and dapD gene as auxotrophic selection system (pCAD_sod::cer::dapD). The aim of this research was to determine the effects of pCAD_sod, pCAD_sod::cer and pCAD_sod::cer::dapD ??®±???? growth profile, sod expression capability and plasmid stability. E. coli cultures carrying each plasmid were grown for 24 hours on Luria Bertani liquid medium with and without ampicillin. Optical density at 600 nm (OD600) was observed to determine cell growth curve. rMnSODSeq amount was observed with SDS-PAGE analysis and dismutation activity was evaluated with zymography. E. coli plasmid stability was determined by calculating the ratio of cells growing on solid LB medium with and without ampicillin. No difference was observed in growth profile of E. coli carrying pCAD_sod and its derivatives. Level of rMnSODSeq protein production was not significantly different at 24 hours and the protein displayed dismutation activity. Plasmid stability at 24 hours for pCAD_sod, pCAD_sod::cer and pCAD_sod::cer::dapD were 100%, 99.7 ± 0.2%, 100%, respectively. Plasmid pCAD_sod and its derivatives do not affect E. coli growth profile, rMnSODSeq expression and plasmid stability. Character of pCAD_sod and its derivatives shows potency of its use as expression vector for recombinant protein production.