Isolation and Characterization of Amylase Produced by CRISPR-Cas 9 Edited LacZ Gene and Unedited Escherichia coli using Potato Peel (Ipomea batata) as Enzyme Source

Abstract

Genome editing, particularly using CRISPR-Cas9, is a powerful tool for manipulating genomes, including Escherichia coli. This study aimed to genetically engineer the lacZ gene in E. coli using CRISPR-Cas9 to assess its role in amylase production during submerged fermentation of sweet potato peels (Ipomoea batatas). Edited and wild-type E. coli were cultured at 37ºC, under pH 6.2, 7.0, and 8.4 conditions, and the resulting amylase was purified using ammonium sulfate. Amylase production was screened using starch as a glucose source, with enzyme characterization performed at varying temperatures and pH levels. CRISPR-Cas9 edited E. coli without the guide RNA (gRNA) and arabinose showed blue colonies, while those with gRNA, Cas9, but without arabinose showed no colonies. Edited E. coli with Cas9, and arabinose, but without gRNA also produced blue colonies. Colonies displayed a white phenotype when subjected to Cas9, gRNA, and arabinose. Gel electrophoresis revealed that E. coli exposed to Cas9 and arabinose had two bands at 650 bp, while blue colonies exposed to Cas9 without gRNA and arabinose showed bands at 1,100 bp. The positive control exhibited three distinct bands, whereas the negative control had none. Amylolytic screening shows similar clear zones of wild-type E. coli and CRISPR-edited E. coli. pH 8.4 provides the most favorable conditions for wild-type E. coli growth and pH 7.0 for the CRISPR-edited E. coli growth during 15 days of fermentation. Temperature and pH assays indicated that both wild-type and CRISPR-edited E. coli showed similar maximum amylase activity at 45ºC and pH 7, with no significant difference in enzyme production. These results suggest that the lacZ gene does not significantly affect amylase production in E. coli.

Keywords

• CRISPR Cas9 gene editing, lacZ gene, E. coli, Potato peel fermentation, Amylase