CRISPR-CAS9 MEDIATED EDITING OF THE LACZ GENE FOR OPTIMIZING LIPASE PRODUCTION AND CHARACTERIZATION IN ENGINEERED ESCHERICHIA COLI USING POTATO PEEL AS A GLUCOSE SOURCE

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ABSTRACT Improper disposal of potato peels harms the environment, wasting nutrient-rich resources that could be used for beneficial enzyme production like lipase. This study aimed to edit the lacZ gene in Escherichia coli using utilizing the CRISPR Cas9 technology. Both edited and unedited E. coli were used for submerged fermentation of potato peels to produce and characterize lipase. The lacZ gene was edited using the CRISPR Cas9 technology, and the efficiency was measured using multiplex PCR and gel electrophoresis. To measure lipolytic activity, olive oil screening was performed. The temperature and pH of the lipase were used to characterize it after partial purification and submerged fermentation at 10°C, 30°C, and 45°C. Blue colonies indicated the lacZ gene was unedited, lacZ gene editing and repair was demonstrated by white colonies, and no colonies demonstrated the editing but not repair of the lacZ gene. Bands at 1,100 bp indicated unedited lacZ gene , while 650 bp showed edited lacZ gene . Increased cell mass was observed at 10°C. CRISPR Cas9 edited E. coli showed a clearer zone than the unedited in the lipase medium. The highest lipase activity from both edited and unedited E. coli was at 35°C, and the lowest at 65°C. Optimal pH was 7, with lowest activity at pH 4. The CRISPR Cas9 edited E. coli demonstrated significantly higher enzyme activities (p<0.05). This study concluded that CRISPR Cas9-mediated lacZ gene editing in E. coli enhances its ability to utilize potato peels, increasing lipase production.

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