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(1 - 8 of 8)
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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Controlling biofilms using synthetic biology approaches
Title
Controlling biofilms using synthetic biology approaches
Creator
Fang, Kuili, Park, Oh-Jin, Hong, Seok Hoon
Date
2020
Description
Bacterial biofilms are formed by the complex but ordered regulation of intra- or inter-cellular communication, environmentally responsive gene...
Show moreBacterial biofilms are formed by the complex but ordered regulation of intra- or inter-cellular communication, environmentally responsive gene expression, and secretion of extracellular polymeric substances. Given the robust nature of bio?lms due to the non-growing nature of bio?lm bacteria and the physical barrier provided by the extracellular matrix, eradicating bio?lms is a very di?cult task to accomplish with conventional antibiotic or disinfectant treatments. Synthetic biology holds substantial promise for controlling bio?lms by improving and expanding existing biological tools, introducing novel functions to the system, and re-conceptualizing gene regulation. This review summarizes synthetic biology approaches used to eradicate bio?lms via protein engineering of bio?lm-related enzymes, utilization of synthetic genetic circuits, and the development of functional living agents. Synthetic biology also enables bene?cial applications of bio?lms through the production of biomaterials and patterning bio?lms with speci?c temporal and spatial structures. Advances in synthetic biology will add novel bio?lm functionalities for future therapeutic, biomanufacturing, and environmental applications.
Sponsorship: NIH-R15AI130988, NSF CBET-1917130
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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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Editorial, "Cell-Free Synthetic Biology": Synthetic Biology Meets Cell-Free Protein Synthesis
Title
Editorial, "Cell-Free Synthetic Biology": Synthetic Biology Meets Cell-Free Protein Synthesis
Creator
Hong, Seok Hoon
Date
2019
Publisher
MDPI
Description
Sponsorship: NIH R15AI130988
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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USING COMPUTATIONAL MOLECULAR MODELING TO STUDY TRANSPORT PROCESSES OF INTEREST IN SEPARATIONS
Title
USING COMPUTATIONAL MOLECULAR MODELING TO STUDY TRANSPORT PROCESSES OF INTEREST IN SEPARATIONS
Creator
Wang,Xiaoyu
Date
2020, 2020
Publisher
ProQuest
Description
Separation processes are widely used in chemical productions. The further development of membrane-based separation processes, compared with...
Show moreSeparation processes are widely used in chemical productions. The further development of membrane-based separation processes, compared with thermal separations, can lead to significant energy savings in chemical process industries. However, the main obstacle of experiments is that many separation processes are not well understood at the fundamental molecular level. In this dissertation, we use computational molecular modeling tools, mainly classical molecular dynamics (MD), to clarify molecular forces and provide detail at a molecular level, which can aid in the understanding of transport process and designing materials for a proposed application. In the first study, we investigated separation of water/alcohol vapor using zeolite membranes. Experimentally, the separation of water/isopropanol (IPA) mixtures shows a dramatic decrease in selectivity due to increase of IPA flux as the feed water concentration decrease when using the sodium A zeolite membrane. We used molecular dynamics simulations to help our experimental collaborators understand these puzzling results. The MD results reveal that the water molecules gather around the defect pores on the zeolite membrane, which stops the IPA from going through the membrane and has a positive effect on separation. Then, we studied the HPLC used to separate chiral drug mixtures. One popular chiral stationary phase, amylose tris(3,5-dimethylphenyl carbamate) (ADMPC), has been investigated using both experimental and computational methods; however, the dynamic nature of the interaction between enantiomers and ADMPC, as well as the solvent effects on the ADMPC-enantiomer interaction, are currently absent from the chiral recognition mechanism. We used MD simulations to model the ADMPC in different solvents to elucidate the chiral recognition mechanism from a new dynamic perspective. The ADMPC is found to hold the left-handed helical structure in both methanol and heptane/IPA (90/10); however, the ADMPC has a more extended average structure in heptane/IPA. We developed a model where the ADMPC atoms were restricted in the MD simulation. To better understand the molecular dynamic chiral recognition that provides the retention factor and the elution order in HPLC, we examined hydrogen bonding lifetimes, and mapped out ring-ring interactions between the drugs and the ADMPC. We discover several MD metrics related to hydrogen-bonding lifetimes and correlate them with HPLC results. One metric provides a prediction of the correct elution order 90%, and the ratios of these quantities for the enantiomers provide linear correlation (0.85 coefficient) with experimental retention factors. In the following study, we presented an improved model wherein multiple ADMPC polymer strands are coated on an amorphous silica slab. Using various MD techniques, we successfully coated ADMPCs onto the surface without losing the structural character of the backbone in the solvent. This model provides more opportunities for chiral molecules interacting with ADMPC, resulting in a better agreement compared with experiment when using the overall average metric. The new model also provides the possibility for drug molecules to interact with two polymer strands simultaneously, which is not possible in the previous single-strand model. For a better understanding of why some metrics are better predictors than others, we used charts of the distribution of hydrogen bonding lifetimes to display the information for various donor-acceptor pairs. The results are more consistent than the previous models and resolves the problematic cases of thalidomide and valsartan. Besides the membrane-based separations, immiscible liquid-liquid equilibrium states were also studied. We successfully predicted results based on MD simulations and showed comparable accuracy with experimental data. This method has applications in liquid-liquid extraction which is widely used in industrial separation process.
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Modeling enantiomeric separations as an interfacial process using amylose tris(3,5-dimethylphenyl carbamate) (ADMPC) polymers coated on amorphous silica
Title
Modeling enantiomeric separations as an interfacial process using amylose tris(3,5-dimethylphenyl carbamate) (ADMPC) polymers coated on amorphous silica
Creator
Wang,Xiaoyu, Jameson, Cynthia, Murad, Sohail
Date
2020, 2020-01-20
Publisher
ACS Langmuir
Description
In the present study, we present a model to predict the chiral separation results for drug enantiomers by ADMPC chiral stationary phase in...
Show moreIn the present study, we present a model to predict the chiral separation results for drug enantiomers by ADMPC chiral stationary phase in high performance liquid chromatography (HPLC) wherein multiple ADMPC polymer strands are coated on an amorphous silica slab. Both reactive and classical MD are used to prepare the surface. Using various MD techniques, we successfully coat ADMPCs onto the surface without losing the structural character of the backbone in the presence of the solvent system. Not only is this model more representative of the polymer surface on a solid support that is encountered by the enantiomers, it also provides more opportunities for chiral molecules interacting with ADMPC, resulting in a better agreement compared with experiment when we use overall average quantities as the metric. In our previous studies, we had used a single polymer strand of amylose tris(3,5-dimethylphenyl carbamate) (ADMPC) in the solvent system. The new model provides the possibility for large drug molecules to interact with two polymer strands at the same instant, which was not possible to model with only a single polymer strand in the solvent. For a better understanding of why some metrics are better predictors than others, we use charts of the distribution of hydrogen bonding lifetimes in this work to display the hydrogen-bonding information for various donor-acceptor pairs that contribute to the interaction events determining the relative retention times for the enantiomers. We also examine the contribution of the ring-ring interactions to the molecular recognition process and ultimately to differential retention of S and R enantiomers. The results using the new model are more consistent than the previous models and resolves the problematic case of two drugs, thalidomide and valsartan.
Sponsorship: The National Science Foundation (CBET 1545560)
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