A programmable CRISPR/Cas9-based phage defense system for Escherichia coli BL21(DE3).

Area of Science:

• Biotechnology
• Microbiology
• Molecular Biology

Background:

• Industrial protein production frequently uses Escherichia coli BL21, which is vulnerable to phage infections during fermentation.
• CRISPR/Cas systems offer programmable defense against phages by targeting specific DNA sequences.

Purpose of the Study:

• To develop and evaluate a CRISPR/Cas9 system for enhancing phage resistance in E. coli BL21.
• To assess the impact of targeted phage genome loci on defense efficacy.
• To optimize CRISPR/Cas9 expression for improved host survival and protein yield under phage pressure.

Main Methods:

• Construction of a CRISPR/Cas9 system targeting multiple sites on the T7 phage genome.
• Testing various expression control modes for the CRISPR/Cas9 components.
• Evaluating bacterial growth (OD) and viable cell counts post-infection.
• Measuring Red Fluorescent Protein (RFP) expression in engineered strains.

Main Results:

• The engineered E. coli BL21 demonstrated increased defense against T7 phage infection.
• Optimized CRISPR/Cas9 expression led to significantly improved optical density (OD) after 4 hours of phage infection, comparable to uninfected controls.
• Engineered strains maintained viable cell counts only one order of magnitude lower than uninfected controls.
• RFP expression in infected engineered strains reached approximately 60% of uninfected controls, outperforming the parent strain.

Conclusions:

• A programmable CRISPR/Cas9 system was successfully constructed to enhance E. coli BL21's phage defense capabilities.
• The developed system creates a more resilient protein expression host, crucial for industrial fermentation.
• This strategy offers a feasible method for protecting industrial E. coli strains from phage contamination.