DEVELOPMENT OF LEAKY STRAIN ESCHERICHIA COLI THROUGH LPP GENE KNOCK-OUT USING LAMBDA-RED RECOMBINEERING

• Main Author: Ni'am Musthofa, Zada
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/78429
• Institution: Institut Teknologi Bandung

E. coli is a microorganism that has been extensively studied and utilized for the production of recombinant proteins due to its fast growth and ease of engineering. One limitation of E. coli in producing recombinant proteins is its inability to secrete them. Recombinant proteins that are secreted outside the cell offer several advantages, such as better protein purity, simplification of downstream processes, and cost savings. To enhance the secretion rate of proteins from the periplasm to the extracellular space, the leaky strain of E. coli can be generated by genome engineering approach via disrupting the gene responsible for the integrity of the outer cell membrane. In this study, we performed lpp gene knocking-out by lambda-red recombineering. This method utilized the lambda-red recombinase enzyme to facilitate the insertion of foreign DNA fragments (knock-out cassette fragments) into the target genome through homologous regions (homologous arms). The process began with the transformation of pSIM18 into an E. coli culture to allow the expression of the lambda-red recombinase. Subsequently, a plasmid carrying the template for the knock-out cassette with a kanamycin antibiotic resistance marker, pKIKO-Kan, was constructed. Knock-out (KO) cassette fragments are produced from this plasmid using extended primers that carry segments of the homologous arm (HA) from the lpp gene for integration into the genome. Electroporation was then performed on the lpp KO cassette fragments, which have been prepared into E. coli at a concentration of 6 ?g/mL (300 ng DNA/50 uL electrocompetent cells) using 1.8 kV, 200 ?, and 25?F. Colonies that successfully grow on agar plates with kanamycin (+ Kan) indicate that the lpp KO cassette has been integrated and has knocked out the lpp gene in the target genome. Further confirmation was carried out on these colonies through colony PCR and sequencing. Colonies on agar plates with kanamycin that were confirmed through colony PCR and sequencing were identified as engineered E. coli strains (?lpp).