KONSTRUKSI PLASMID pCAD-cer2-dapD PADA ESCHERICHIA COLI

• Main Author: Bahar, Zulhaerana
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/44591
• Institution: Institut Teknologi Bandung

One of the biopharmaceutical products is recombinant therapeutic proteins. Production of recombinant therapeutic proteins is influenced by plasmid copy number and plasmid stability carrying the encoded gene. Plasmids with low copy number, stable, and carrying antibiotic free selection system are preferred for the production of therapeutic proteins. Plasmid stability can be increased by the addition of cer DNA fragment. One of the antibiotic-free selection systems that can be used is lysine auxotroph based system. dapD gene encoding DapD enzyme that important in lysine biosynthesis pathway, in which deletions and mutations of this gene are lethal to bacteria. Hence, it can be used for auxotrophic selection system. In previous study, pCAD-cer plasmid has been successfully constructed, which was carrying sod expression cassette, cer fragment and ori of pBR322. dapD gene has been isolated from Escherichia coli in previous study and was mutated to eliminate NdeI restriction enzyme site. This study aims to insert dapD gene into pCAD-cer to obtain low copy number plasmid and can be selected by auxotrophic selection system. Wild type and mutant dapD gene were each inserted into pCAD-cer and was confirmed by migration, restriction enzyme using NdeI, PCR, and nucleotide sequencing analyses. The constructed plasmid, pCAD-cer-dapD was shown to express active SOD of 22 kDa and DapD protein of 31 kDa. The pCAD-cer2-dapD can be used further as expression vector with lysine auxotrophic selection system.