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Optimizing Cell-Free Protein Synthesis for Increased Yield and Activity of Colicins
Title
Optimizing Cell-Free Protein Synthesis for Increased Yield and Activity of Colicins
Creator
Jin, Xing, Kightlinger, Weston, Hong, Seok Hoon
Date
2019
Publisher
MDPI
Description
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
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Engineering Escherichia coli to produce and secrete colicins for rapid and selective biofilm cell killing
Title
Engineering Escherichia coli to produce and secrete colicins for rapid and selective biofilm cell killing
Creator
Jin, Xing, An, Sungjun, Kightlinger, Weston, Zhou, Jiacheng, Hong, Seok Hoon
Date
2021
Description
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.
Sponsorship: NIH-R15AI130988
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Incorporation of non-standard amino acids into proteins: challenges, recent achievements, and emerging applications
Title
Incorporation of non-standard amino acids into proteins: challenges, recent achievements, and emerging applications
Creator
Jin, Xing, Park Oh-Jin, Hong, Seok Hoon
Date
2019
Description
The natural genetic code only allows for 20 standard amino acids in protein translation, but genetic code reprogramming enables the...
Show moreThe natural genetic code only allows for 20 standard amino acids in protein translation, but genetic code reprogramming enables the incorporation of non-standard amino acids (NSAAs). Proteins containing NSAAs provide enhanced or novel properties and open diverse applications. With increased attention to the recent advancements in synthetic biology, various improved and novel methods have been developed to incorporate single and multiple distinct NSAAs into proteins. However, various challenges remain in regard to NSAA incorporation, such as low yield and misincorporation. In this review, we summarize the recent efforts to improve NSAA incorporation by utilizing orthogonal translational system optimization, cell-free protein synthesis, genomically recoded organisms, artificial codon boxes, quadruplet codons, and orthogonal ribosomes, before closing with a discussion of the emerging applications of NSAA incorporation.
Sponsorship: NIH R15AI130988
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Undecanoic Acid, Lauric Acid, and N-Tridecanoic Acid Inhibit Escherichia coli Persistence and Biofilm Formation
Title
Undecanoic Acid, Lauric Acid, and N-Tridecanoic Acid Inhibit Escherichia coli Persistence and Biofilm Formation
Creator
Jin, Xing, Zhou, Jiacheng, Richey, Gabriella, Wang, Mengya, Hong, Sung Min Choi, Hong, Seok Hoon
Date
2021
Description
Persister cell formation and biofilms of pathogens are extensively involved in the development of chronic infectious diseases. Eradicating...
Show morePersister cell formation and biofilms of pathogens are extensively involved in the development of chronic infectious diseases. Eradicating persister cells is challenging, owing to their tolerance to conventional antibiotics, which cannot kill cells in a metabolically dormant state. A high frequency of persisters in biofilms makes inactivating biofilm cells more difficult, because the biofilm matrix inhibits antibiotic penetration. Fatty acids may be promising candidates as antipersister or antibiofilm agents, because some fatty acids exhibit antimicrobial effects. We previously reported that fatty acid ethyl esters effectively inhibit Escherichia coli persister formation by regulating an antitoxin. In this study, we screened a fatty acid library consisting of 65 different fatty acid molecules for altered persister formation. We found that undecanoic acid, lauric acid, and N-tridecanoic acid inhibited E. coli BW25113 persister cell formation by 25-, 58-, and 44-fold, respectively. Similarly, these fatty acids repressed persisters of enterohemorrhagic E. coli EDL933. These fatty acids were all medium-chain saturated forms. Furthermore, the fatty acids repressed Enterohemorrhagic E. coli (EHEC) biofilm formation (for example, by 8-fold for lauric acid) without having antimicrobial activity. This study demonstrates that medium-chain saturated fatty acids can serve as antipersister and antibiofilm agents that may be applied to treat bacterial infections.
Sponsorship: NIH-R15AI130988; NSF REU-1757989
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