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- Optimizing Cell-Free Protein Synthesis for Increased Yield and Activity of Colicins
- Jin, Xing, Kightlinger, Weston, Hong, Seok Hoon
Colicins are antimicrobial proteins produced by Escherichia coli that hold great promise as viable complements or alternatives to antibiotics....
Show moreColicins are antimicrobial proteins produced by Escherichia coli that hold great promise as viable complements or alternatives to antibiotics. Cell-free protein synthesis (CFPS) is a useful production platform for toxic proteins because it eliminates the need to maintain cell viability, a common problem in cell-based production. Previously, we demonstrated that colicins produced by CFPS based on crude Escherichia coli lysates are e?ective in eradicating antibiotic-tolerant bacteria known as persisters. However, we also found that some colicins have poor solubility or low cell-killing activity. In this study, we improved the solubility of colicin M from 16% to nearly 100% by producing it in chaperone-enriched E. coli extracts, resulting in enhanced cell-killing activity. We also improved the cytotoxicity of colicin E3 by adding or co-expressing the E3 immunity protein during the CFPS reaction, suggesting that the E3 immunity protein enhances colicin E3 activity in addition to protecting the host strain. Finally, we con?rmed our previous ?nding that active colicins can be rapidly synthesized by observing colicin E1 production over time in CFPS. Within three hours of CFPS incubation, colicin E1 reached its maximum production yield and maintained high cytotoxicity during longer incubations up to 20 h. Taken together, our ?ndings indicate that colicin production can be easily optimized for improved solubility and activity using the CFPS platform.
Sponsorship: NIH R15AI130988
- Engineering Escherichia coli to produce and secrete colicins for rapid and selective biofilm cell killing
- Jin, Xing, An, Sungjun, Kightlinger, Weston, Zhou, Jiacheng, Hong, Seok Hoon
Bacterial biofilms are associated with chronic infectious diseases and are highly resistant to conventional antibiotics. Antimicrobial...
Show moreBacterial biofilms are associated with chronic infectious diseases and are highly resistant to conventional antibiotics. Antimicrobial bacteriocins are alternatives to conventional antibiotics and are characterized by unique cell-killing mechanisms, including pore formation on cell membranes, nuclease activity, and cell wall synthesis inhibition. Here, we used cell-free protein synthesis to rapidly evaluate the antibiofilm activities of colicins E1, E2, and E3. We found that E2 (with DNase activity) most effectively killed target biofilm cells (i.e., the K361 strain) while leaving nontargeted biofilms intact. We then engineered probiotic Escherichia coli microorganisms with genetic circuits to controllably synthesize and secrete colicin E2, which successfully inhibited biofilms and killed preformed indicator biofilms. Our findings suggest that colicins rapidly and selectively kill target biofilm cells in multispecies biofilms and demonstrate the potential of using microorganisms engineered to produce antimicrobial colicin proteins as live therapeutic strategies to treat biofilm-associated infections.