MODIFICATION OF NDEI RESTRICTION SITE IN TETRAHYDRODIPICOLINATE N-SUCCINYLTRANSFERASE (DAPD) GENE OF ESCHERICHIA COLI BL21(DE3)

• Main Author: Yuliana, Yurike
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/79006
• Institution: Institut Teknologi Bandung

Tetrahydrodipicolinate N-succinyltransferase (dapD) gene is one of genes which can be inserted into a plasmid for auxotroph selection system. This gene acts as transformant selection marker for replacing antibiotics selection system. In the previous research, a plasmid with autoinduction system (pCAD) has been successfully constructed. Antibiotic selection system used in pCAD will be replaced by dapD auxotroph selection system. Ndel restriction site is commonly used in Multiple Cloning Site (MCS), therefore Ndel restriction site in dapD gene must be modified. This research aimed to isolate dapD gene from Escherichia coli genomes, to clone the gene to pGEM-T easy vector and to modify Ndel restriction site in dapD gene. Therefore, the dapD gene is readily used for antibioticfree selection system in pCAD. Isolation of dapD gene was performed by Polymerase Chain Reaction (PCR) then dapD gene was inserted into pGEM-T easy vector to form pGEM dap. This plasmid was confirmed by several analyses. The modification of Ndel restriction site in pGEM dap was performed by Site-Directed Mutagenesis and the result was confirmed by several analyses. This research showed that the dapD gene was isolated and cloned to pGEM-T easy vector, and Ndel restriction site in dapD gene was modified. These results were confirmed by migration, restriction, PCR, and DNA sequencing analyses.