Gene Editing Using E. coli 1. Plasmids Plasmids are small, circular DNA molecules used as vectors to introduce foreign genes into E. coli. Key features of plasmids include: • Origin of replication (ori ): Ensures plasmid replication within E. coli. • Selectable markers: Genes such as ampR (ampicillin resistance) allow for selection of transformed cells. • Multiple cloning sites (MCS): Contains recognition sites for restriction enzymes. 2. Restriction Enzymes Restriction enzymes (endonucleases) cut DNA at specific sequences, enabling the insertion of target genes into plasmids. 3. DNA Ligase DNA ligase joins DNA fragments, sealing nicks in the sugar-phosphate backbone. 4. CRISPR-Cas9 The CRISPR-Cas9 system enables precise gene editing. The Cas9 protein creates doublestrand breaks guided by a custom-designed single-guide RNA (sgRNA). Gene Editing Techniques in E. coli 1. Transformation Transformation involves introducing plasmids into E. coli cells. Methods include: 1 • Chemical transformation: E. coli cells are made competent with calcium chloride and heat-shocked to take up DNA. • Electroporation: High-voltage pulses create pores in the cell membrane for DNA entry. 2. Homologous Recombination Homologous recombination allows for precise gene replacement or deletion by exchanging segments of DNA with homologous sequences. 3. Site-Directed Mutagenesis Site-directed mutagenesis introduces specific changes in a gene, such as point mutations, deletions, or insertions. Applications of Gene Editing in E. coli 1. Protein Production E. coli is used to express recombinant proteins for research, industrial, and therapeutic purposes. 2. Metabolic Engineering Metabolic pathways in E. coli can be modified to enhance the production of biofuels, bioplastics, and other valuable compounds. 3. Functional Genomics Gene editing enables the study of gene function by creating knockouts or overexpressing specific genes. Limitations and Challenges • Off-target effects in CRISPR-Cas9 editing. • Limited efficiency of homologous recombination. 2 • Challenges in expressing eukaryotic proteins in E. coli. 3