Michael Christopher Jewett

All Publications

• Design-driven optimization of low-cost reagent formulations for reproducible and high-yielding cell-free gene expression. Nature communications Olsen, M. L., Copeland, C. E., Sundberg, C. A., Aw, R., Shaver, Z. M., Rao, G., Swartz, J. R., Karim, A. S., Jewett, M. C. 2026

  Abstract

  Access to recombinant proteins is vital in basic science and biotechnology research. Cell-free gene expression systems provide one approach to address this need, but widespread utilization remains limited by the cost, complexity, and inconsistency of current platforms. To address these limitations, we carry out a multi-dimensional definitive screening design to reduce the number of reagent components and remove costly secondary energy substrates. From 1,231 different reagent formulations, we discover a simple and reproducible system based on 12 components. The optimized reagent formulation can produce 2.4 ± 0.3 g/L of protein product at the 15-µL scale (~$60/gprotein) and 3.7 ± 0.2 g/L (~$39/gprotein) at the 4-mL scale with oxygen supplementation. This provides an average 95% reduction in cost over previous cell-free reagent formulations. We further show that the optimized reagent formulation can produce nucleoside triphosphates from nitrogenous bases and ribose and that it is robust to failure across batches of cell lysates, users/locations, and in the synthesis of more than 20 different proteins. For example, we demonstrate the production of fifteen therapeutically relevant products, including full-length aglycosylated monoclonal antibodies. We anticipate that our optimized reagent formulation will democratize the use of cell-free systems for protein manufacturing and synthetic biology applications.

  View details for DOI 10.1038/s41467-026-69605-8

  View details for PubMedID 41786720

• A synthetic cell-free pathway for biocatalytic upgrading of formate from electrochemically reduced CO₂ NATURE CHEMICAL ENGINEERING Landwehr, G. M., Vogeli, B., Tian, C., Singal, B., Zolkin, K., Martinez, I., Gupta, A., Lion, R., Sargent, E. H., Karim, A. S., Jewett, M. C. 2025

  View details for DOI 10.1038/s44286-025-00315-6

  View details for Web of Science ID 001644220000001

• Active learning-guided optimization of cell-free biosensors for lead testing in drinking water. bioRxiv : the preprint server for biology Wang, B. M., Chiang, N., Ekas, H. M., Brown, D. M., Dildine, G., Lucci, T. J., Feng, S., Bly, V., Gaillard, J. F., Lucks, J. B., Karim, A. S., Shukla, D., Jewett, M. C. 2025

  Abstract

  Point-of-use diagnostics based on allosteric transcription factors (aTFs) are promising tools for environmental monitoring and human health. However, biosensors relying on natural aTFs rarely exhibit the sensitivity and selectivity needed for real-world applications, and traditional directed evolution struggles to optimize multiple biosensor properties at once. To overcome these challenges, we develop a multi-objective, machine learning (ML)-guided cell-free gene expression workflow for engineering aTF-based biosensors. Our approach rapidly generates high-quality sequence-to-function data, which we transform into an augmented paired dataset to train an ML model using directional labels that capture how aTF mutations alter performance. We apply our workflow to engineer the aTF PbrR as a point-of-use diagnostic for lead contamination in water. We tune the sensitivity of PbrR to sense at the U.S. Environmental Protection Agency (EPA) action level for lead and modify the selectivity away from zinc, a common metal found in water supplies. Finally, we show that the engineered PbrR functions in freeze-dried cell-free reactions, enabling a diagnostic capable of detecting lead in drinking water down to ~5.7 ppb. Our ML-driven, multi-objective framework-powered by directional tokens-can generalize to other biosensors and proteins, accelerating the development of synthetic biology tools for biotechnology applications.

  View details for DOI 10.1101/2025.08.20.671382

  View details for PubMedID 40894645

  View details for PubMedCentralID PMC12393547

• Accelerated enzyme engineering by machine-learning guided cell-free expression. Nature communications Landwehr, G. M., Bogart, J. W., Magalhaes, C., Hammarlund, E. G., Karim, A. S., Jewett, M. C. 2025; 16 (1): 865

  Abstract

  Enzyme engineering is limited by the challenge of rapidly generating and using large datasets of sequence-function relationships for predictive design. To address this challenge, we develop a machine learning (ML)-guided platform that integrates cell-free DNA assembly, cell-free gene expression, and functional assays to rapidly map fitness landscapes across protein sequence space and optimize enzymes for multiple, distinct chemical reactions. We apply this platform to engineer amide synthetases by evaluating substrate preference for 1217 enzyme variants in 10,953 unique reactions. We use these data to build augmented ridge regression ML models for predicting amide synthetase variants capable of making 9 small molecule pharmaceuticals. Over these nine compounds, ML-predicted enzyme variants demonstrate 1.6- to 42-fold improved activity relative to the parent. Our ML-guided, cell-free framework promises to accelerate enzyme engineering by enabling iterative exploration of protein sequence space to build specialized biocatalysts in parallel.

  View details for DOI 10.1038/s41467-024-55399-0

  View details for PubMedID 39833164

  View details for PubMedCentralID 5892708

• Carbon-negative production of acetone and isopropanol by gas fermentation at industrial pilot scale NATURE BIOTECHNOLOGY Liew, F., Nogle, R., Abdalla, T., Rasor, B. J., Canter, C., Jensen, R. O., Wang, L., Strutz, J., Chirania, P., De Tissera, S., Mueller, A. P., Ruan, Z., Gao, A., Tran, L., Engle, N. L., Bromley, J. C., Daniell, J., Conrado, R., Tschaplinski, T. J., Giannone, R. J., Hettich, R. L., Karim, A. S., Simpson, S. D., Brown, S. D., Leang, C., Jewett, M. C., Kopke, M. 2022; 40 (3): 335-+

  Abstract

  Many industrial chemicals that are produced from fossil resources could be manufactured more sustainably through fermentation. Here we describe the development of a carbon-negative fermentation route to producing the industrially important chemicals acetone and isopropanol from abundant, low-cost waste gas feedstocks, such as industrial emissions and syngas. Using a combinatorial pathway library approach, we first mined a historical industrial strain collection for superior enzymes that we used to engineer the autotrophic acetogen Clostridium autoethanogenum. Next, we used omics analysis, kinetic modeling and cell-free prototyping to optimize flux. Finally, we scaled-up our optimized strains for continuous production at rates of up to ~3 g/L/h and ~90% selectivity. Life cycle analysis confirmed a negative carbon footprint for the products. Unlike traditional production processes, which result in release of greenhouse gases, our process fixes carbon. These results show that engineered acetogens enable sustainable, high-efficiency, high-selectivity chemicals production. We expect that our approach can be readily adapted to a wide range of commodity chemicals.

  View details for DOI 10.1038/s41587-021-01195-w

  View details for Web of Science ID 000758993700004

  View details for PubMedID 35190685

  View details for PubMedCentralID 7356534

• On-demand biomanufacturing of protective conjugate vaccines SCIENCE ADVANCES Stark, J. C., Jaroentomeechai, T., Moeller, T. D., Hershewe, J. M., Warfel, K. F., Moricz, B. S., Martini, A. M., Dubner, R. S., Hsu, K. J., Stevenson, T. C., Jones, B. D., DeLisa, M. P., Jewett, M. C. 2021; 7 (6)

  Abstract

  Conjugate vaccines are among the most effective methods for preventing bacterial infections. However, existing manufacturing approaches limit access to conjugate vaccines due to centralized production and cold chain distribution requirements. To address these limitations, we developed a modular technology for in vitro conjugate vaccine expression (iVAX) in portable, freeze-dried lysates from detoxified, nonpathogenic Escherichia coli. Upon rehydration, iVAX reactions synthesize clinically relevant doses of conjugate vaccines against diverse bacterial pathogens in 1 hour. We show that iVAX-synthesized vaccines against Francisella tularensis subsp. tularensis (type A) strain Schu S4 protected mice from lethal intranasal F. tularensis challenge. The iVAX platform promises to accelerate development of new conjugate vaccines with increased access through refrigeration-independent distribution and portable production.

  View details for DOI 10.1126/sciadv.abe9444

  View details for Web of Science ID 000615369000039

  View details for PubMedID 33536221

  View details for PubMedCentralID PMC7857678

• In vitro prototyping and rapid optimization of biosynthetic enzymes for cell design. Nature chemical biology Karim, A. S., Dudley, Q. M., Juminaga, A., Yuan, Y., Crowe, S. A., Heggestad, J. T., Garg, S., Abdalla, T., Grubbe, W. S., Rasor, B. J., Coar, D. N., Torculas, M., Krein, M., Liew, F. E., Quattlebaum, A., Jensen, R. O., Stuart, J. A., Simpson, S. D., Köpke, M., Jewett, M. C. 2020; 16 (8): 912-919

  Abstract

  The design and optimization of biosynthetic pathways for industrially relevant, non-model organisms is challenging due to transformation idiosyncrasies, reduced numbers of validated genetic parts and a lack of high-throughput workflows. Here we describe a platform for in vitro prototyping and rapid optimization of biosynthetic enzymes (iPROBE) to accelerate this process. In iPROBE, cell lysates are enriched with biosynthetic enzymes by cell-free protein synthesis and then metabolic pathways are assembled in a mix-and-match fashion to assess pathway performance. We demonstrate iPROBE by screening 54 different cell-free pathways for 3-hydroxybutyrate production and optimizing a six-step butanol pathway across 205 permutations using data-driven design. Observing a strong correlation (r = 0.79) between cell-free and cellular performance, we then scaled up our highest-performing pathway, which improved in vivo 3-HB production in Clostridium by 20-fold to 14.63 ± 0.48 g l-1. We expect iPROBE to accelerate design-build-test cycles for industrial biotechnology.

  View details for DOI 10.1038/s41589-020-0559-0

  View details for PubMedID 32541965

  View details for PubMedCentralID 373084

• BioBits (TM) Bright: A fluorescent synthetic biology education kit SCIENCE ADVANCES Stark, J. C., Huang, A., Nguyen, P. Q., Dubner, R. S., Hsu, K. J., Ferrante, T. C., Anderson, M., Kanapskyte, A., Mucha, Q., Packett, J. S., Patel, P., Patel, R., Qaq, D., Zondor, T., Burke, J., Martinez, T., Miller-Berry, A., Puppala, A., Reichert, K., Schmid, M., Brand, L., Hill, L. R., Chellaswamy, J. F., Faheem, N., Fetherling, S., Gong, E., Gonzalzles, E., Granito, T., Koritsaris, J., Binh Nguyen, Ottman, S., Palffy, C., Patel, A., Skweres, S., Slaton, A., Woods, T., Donghia, N., Pardee, K., Collins, J. J., Jewett, M. C. 2018; 4 (8): eaat5107

  Abstract

  Synthetic biology offers opportunities for experiential educational activities at the intersection of the life sciences, engineering, and design. However, implementation of hands-on biology activities in classrooms is challenging because of the need for specialized equipment and expertise to grow living cells. We present BioBits™ Bright, a shelf-stable, just-add-water synthetic biology education kit with easy visual outputs enabled by expression of fluorescent proteins in freeze-dried, cell-free reactions. We introduce activities and supporting curricula for teaching the central dogma, tunable protein expression, and design-build-test cycles and report data generated by K-12 teachers and students. We also develop inexpensive incubators and imagers, resulting in a comprehensive kit costing

  View details for DOI 10.1126/sciadv.aat5107

  View details for Web of Science ID 000443498100062

  View details for PubMedID 30083609

  View details for PubMedCentralID PMC6070313

• Cell-free protein synthesis from genomically recoded bacteria enables multisite incorporation of noncanonical amino acids NATURE COMMUNICATIONS Martin, R. W., Des Soye, B. J., Kwon, Y., Kay, J., Davis, R. G., Thomas, P. M., Majewska, N. I., Chen, C. X., Marcum, R. D., Weiss, M., Stoddart, A. E., Amiram, M., Charna, A., Patel, J. R., Isaacs, F. J., Kelleher, N. L., Hong, S., Jewett, M. C. 2018; 9: 1203

  Abstract

  Cell-free protein synthesis has emerged as a powerful approach for expanding the range of genetically encoded chemistry into proteins. Unfortunately, efforts to site-specifically incorporate multiple non-canonical amino acids into proteins using crude extract-based cell-free systems have been limited by release factor 1 competition. Here we address this limitation by establishing a bacterial cell-free protein synthesis platform based on genomically recoded Escherichia coli lacking release factor 1. This platform was developed by exploiting multiplex genome engineering to enhance extract performance by functionally inactivating negative effectors. Our most productive cell extracts enabled synthesis of 1,780 ± 30 mg/L superfolder green fluorescent protein. Using an optimized platform, we demonstrated the ability to introduce 40 identical p-acetyl-L-phenylalanine residues site specifically into an elastin-like polypeptide with high accuracy of incorporation ( ≥ 98%) and yield (96 ± 3 mg/L). We expect this cell-free platform to facilitate fundamental understanding and enable manufacturing paradigms for proteins with new and diverse chemistries.

  View details for DOI 10.1038/s41467-018-03469-5

  View details for Web of Science ID 000428165400002

  View details for PubMedID 29572528

  View details for PubMedCentralID PMC5865108

• Single-pot glycoprotein biosynthesis using a cell-free transcription-translation system enriched with glycosylation machinery. Nature communications Jaroentomeechai, T. n., Stark, J. C., Natarajan, A. n., Glasscock, C. J., Yates, L. E., Hsu, K. J., Mrksich, M. n., Jewett, M. C., DeLisa, M. P. 2018; 9 (1): 2686

  Abstract

  The emerging discipline of bacterial glycoengineering has made it possible to produce designer glycans and glycoconjugates for use as vaccines and therapeutics. Unfortunately, cell-based production of homogeneous glycoproteins remains a significant challenge due to cell viability constraints and the inability to control glycosylation components at precise ratios in vivo. To address these challenges, we describe a novel cell-free glycoprotein synthesis (CFGpS) technology that seamlessly integrates protein biosynthesis with asparagine-linked protein glycosylation. This technology leverages a glyco-optimized Escherichia coli strain to source cell extracts that are selectively enriched with glycosylation components, including oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs). The resulting extracts enable a one-pot reaction scheme for efficient and site-specific glycosylation of target proteins. The CFGpS platform is highly modular, allowing the use of multiple distinct OSTs and structurally diverse LLOs. As such, we anticipate CFGpS will facilitate fundamental understanding in glycoscience and make possible applications in on demand biomanufacturing of glycoproteins.

  View details for DOI 10.1038/s41467-018-05110-x

  View details for PubMedID 30002445

  View details for PubMedCentralID PMC6043479

• Protein synthesis by ribosomes with tethered subunits NATURE Orelle, C., Carlson, E. D., Szal, T., Florin, T., Jewett, M. C., Mankin, A. S. 2015; 524 (7563): 119-U289

  Abstract

  The ribosome is a ribonucleoprotein machine responsible for protein synthesis. In all kingdoms of life it is composed of two subunits, each built on its own ribosomal RNA (rRNA) scaffold. The independent but coordinated functions of the subunits, including their ability to associate at initiation, rotate during elongation, and dissociate after protein release, are an established model of protein synthesis. Furthermore, the bipartite nature of the ribosome is presumed to be essential for biogenesis, since dedicated assembly factors keep immature ribosomal subunits apart and prevent them from translation initiation. Free exchange of the subunits limits the development of specialized orthogonal genetic systems that could be evolved for novel functions without interfering with native translation. Here we show that ribosomes with tethered and thus inseparable subunits (termed Ribo-T) are capable of successfully carrying out protein synthesis. By engineering a hybrid rRNA composed of both small and large subunit rRNA sequences, we produced a functional ribosome in which the subunits are covalently linked into a single entity by short RNA linkers. Notably, Ribo-T was not only functional in vitro, but was also able to support the growth of Escherichia coli cells even in the absence of wild-type ribosomes. We used Ribo-T to create the first fully orthogonal ribosome-messenger RNA system, and demonstrate its evolvability by selecting otherwise dominantly lethal rRNA mutations in the peptidyl transferase centre that facilitate the translation of a problematic protein sequence. Ribo-T can be used for exploring poorly understood functions of the ribosome, enabling orthogonal genetic systems, and engineering ribosomes with new functions.

  View details for DOI 10.1038/nature14862

  View details for Web of Science ID 000359002300044

  View details for PubMedID 26222032

• Impact of Process Interruptions in the Production of Lysates for Cell-Free Expression Systems. Biotechnology and bioengineering Rhea, K. A., Walters, E. N., Zacharko, J. L., Seergae, M. J., Laws, T. R., Maishman, T. C., Choi, Y. N., Lazar, J. T., Karim, A., Kightlinger, W., Jewett, M. C., Lux, M. W. 2026

  Abstract

  Cell-free gene expression systems offer cell-like functionalities outside the confines of the cell, garnering increasing interest for applications from biomanufacturing to sensing. As applications expand, the need to implement economically scaled processes to produce cellular lysates grows. The protocols to produce these cellular lysates are complex, and the impact of altering many of the process variables remains understudied. Here, we set out to evaluate the effect of extended incubations at several points in the extract preparation process with the goal of identifying breakpoints that would enable flexibility in process implementation. As a model, we prepared lysates from 50 L cultures instead of typical 1 L volumes. We produced 72 lysates, 36 that were incubated overnight before and after culture centrifugation, and 36 that were incubated with and without a run-off reaction, each across different temperatures. We found that incubations before and after culture centrifugation substantially increased variability between culture replicates but did not reduce cell-free protein synthesis activity, contrary to conventional wisdom that materials should be kept cold as much as possible throughout the process. We also observed that omitting the run-off reaction reduced yields but resulted in lysates that were robust to incubation up to room temperature overnight. When a run-off reaction was included, activity dropped both as a function of duration and temperature, and the overall variability increased. Our work offers potential options for flexibility in implementing lysate production processes and motivates further investigation into how key processing steps relate to cell-free expression activity.

  View details for DOI 10.1002/bit.70199

  View details for PubMedID 41936042

• Topological reprogramming transforms an integral membrane oligosaccharyltransferase into a water-soluble glycosylation catalyst. bioRxiv : the preprint server for biology Kwon, Y. H., Mihaljević, L., Kim, K., Kim, D. E., Donahue, T. C., Bidstrup, E. J., Bandi, C. K., Sotomayor, B., Hulbert, S. W., Myers, K. A., Tian, A., Culpepper, M., Mizrachi, D., Jaroentomeechai, T., Clausen, H., Jewett, M. C., Baker, D., DeLisa, M. P. 2026

  Abstract

  Glycosyltransferases (GTs) catalyze the formation of new glycosidic bonds and thus are vital for synthesizing nature's vast repertoire of glycans and glycoconjugates and for engineering glycan-related medicines and materials. However, obtaining detailed structural and functional insights for the >750,000 known GTs is limited by difficulties associated with their efficient recombinant expression. Members of the GT-C fold, in particular, pose the most significant expression challenges due to the integration and folding requirements of their multiple membrane-spanning regions. Here, we address this challenge by engineering water-soluble variants of an archetypal GT-C fold enzyme, namely the oligosaccharyltransferase PglB from Campylobacter jejuni (CjPglB), which possesses 13 hydrophobic transmembrane helices. To render CjPglB water-soluble, we leveraged two advanced protein engineering methods: one that is universal called SIMPLEx (solubilization of IMPs with high levels of expression) and the other that is custom tailored called WRAPs (water-soluble RFdiffused amphipathic proteins). Each approach was able to transform CjPglB into a water-soluble enzyme that could be readily expressed in the cytoplasm of Escherichia coli cells at yields in the 3-6 mg/L range. Importantly, solubilization was achieved without the need for detergents and with retention of catalytic function. Collectively, our findings demonstrate that both SIMPLEx and WRAPs are promising platforms for advancing the molecular characterization of even the most structurally complex GTs, while also enabling broader GT-mediated glycosylation capabilities within synthetic glycobiology applications.

  View details for DOI 10.64898/2026.01.30.702934

  View details for PubMedID 41659604

  View details for PubMedCentralID PMC12879670

• Design of solubly expressed miniaturized SMART MHCs. Proceedings of the National Academy of Sciences of the United States of America White, W. L., Bai, H., Kim, C. J., Jude, K. M., Sun, R., Guerrero, L., Han, X., Chen, X., Chaudhuri, A., Bonzanini, J. E., Sun, Y., Onwuka, A. E., Wang, N., Wang, C., Nygren, P. Å., Li, X., Goreshnik, I., Allen, A., Levine, P. M., Kueh, H. Y., Jewett, M. C., Sgourakis, N. G., Achour, A., Garcia, K. C., Baker, D. 2026; 123 (1): e2505932123

  Abstract

  The precise recognition of specific peptide-major histocompatibility complex (pMHC) complexes by T cell receptors (TCRs) plays a key role in infectious disease, cancer, and autoimmunity. A critical step in many immunobiological studies is the identification of T cells expressing TCRs specific to a given pMHC antigen. However, the intrinsic instability of empty class-I MHCs limits their soluble expression in Escherichia coli and makes it very difficult to characterize even a small fraction of possible pMHC/TCR interactions. To overcome this limitation, we designed small proteins which buttress the peptide binding groove of class I MHCs, replacing β2-microglobulin (β2m) and the heavy chain α3 domain, and enable soluble and partially soluble expression in E. coli of H-2Db and A*02:01, respectively. We demonstrate that these soluble, monomeric, antigen-receptive, truncated (SMART) MHCs retain both peptide- and TCR-binding specificity and that peptide-bound structures of both allomorphs are similar to their full-length, native counterparts. With extension to the majority of HLA alleles, SMART MHCs should be broadly useful for probing the T cell repertoire in approaches ranging from yeast display to T cell staining.

  View details for DOI 10.1073/pnas.2505932123

  View details for PubMedID 41481462

• Active learning-guided optimization of cell-free biosensors for lead testing in drinking water. Nature communications Wang, B. M., Chiang, N., Ekas, H. M., Brown, D. M., Dildine, G., Lucci, T. J., Feng, S., Bly, V., Gaillard, J. F., Lucks, J. B., Karim, A. S., Shukla, D., Jewett, M. C. 2025

  Abstract

  Point-of-use diagnostics based on allosteric transcription factors (aTFs) are promising tools for environmental monitoring and human health. However, biosensors relying on natural aTFs rarely exhibit the sensitivity and selectivity needed for real-world applications, and traditional directed evolution struggles to optimize multiple biosensor properties at once. To overcome these challenges, we develop a multi-objective, machine learning (ML)-guided cell-free gene expression workflow for engineering aTF-based biosensors. Our approach rapidly generates high-quality sequence-to-function data, which we transform into an augmented paired dataset to train an ML model using directional labels that capture how aTF mutations alter performance. We apply our workflow to engineer the aTF PbrR as a point-of-use diagnostic for lead contamination in water. We tune the sensitivity of PbrR to sense at the U.S. Environmental Protection Agency (EPA) action level for lead and modify the selectivity away from zinc, a common metal found in water supplies. Finally, we show that the engineered PbrR functions in freeze-dried cell-free reactions, enabling a diagnostic capable of detecting lead in drinking water down to ~5.7 ppb. Our ML-driven, multi-objective framework powered by directional tokens can generalize to other biosensors and proteins, accelerating the development of synthetic biology tools for biotechnology applications.

  View details for DOI 10.1038/s41467-025-66964-6

  View details for PubMedID 41422091

• A Scalable Cell-Free Manufacturing Platform for Two-Step Bioproduction of Immunogenic Conjugate Vaccines. ACS synthetic biology Wong, D. A., Aw, R., Hulbert, S. W., Qin, Y., Shaver, Z. M., Myers, K. A., Karim, A. S., DeLisa, M. P., Jewett, M. C. 2025

  Abstract

  Rapid and decentralized vaccine production is essential to ensure global preparedness against emerging and re-emerging infectious diseases. Cell-free gene expression systems, which can be freeze-dried for long-term storage and reactivated for point-of-use synthesis, offer a promising solution to address this need. However, scalable cell-free production of conjugate vaccines─highly effective tools against bacterial infections─has been hindered by low yields and inefficient glycosylation. To address these challenges, we developed a modular, cell-free platform for the synthesis and purification of conjugate vaccines. By decoupling cell-free protein expression from in vitro glycosylation in a two-step approach, we achieved >85% glycosylation efficiency and up to ∼450 mg/L of glycoprotein. We applied this platform to manufacture protein-polysaccharide conjugates composed of vaccine carrier proteins covalently modified with polysaccharide antigens from enterotoxigenic Escherichia coli O78 and Streptococcus pneumoniae serotype 4. Our workflow produced conjugate vaccine candidates in under 5 days with >87% product purity and low endotoxin levels suitable for preclinical evaluation. Immunization of mice with the pneumococcal conjugate vaccine induced a strong IgG response against S. pneumoniae serotype 4 capsular polysaccharide, confirming the immunogenicity of the conjugate. We anticipate that this cell-free platform will advance efforts in decentralized manufacturing and rapid response to bacterial disease threats.

  View details for DOI 10.1021/acssynbio.5c00569

  View details for PubMedID 41261044

• LDBT instead of DBTL: combining machine learning and rapid cell-free testing. Nature communications Clark-ElSayed, A., Harrison, I. M., Olsen, M. L., Lazar, J. T., Jewett, M. C., Ellington, A. D. 2025; 16 (1): 9782

  View details for DOI 10.1038/s41467-025-65281-2

  View details for PubMedID 41193493

  View details for PubMedCentralID 2782888

• Engineered orthogonal translation systems from metagenomic libraries expand the genetic code. bioRxiv : the preprint server for biology Seki, K., Nguyen, M. T., Penev, P. I., Banfield, J. F., Isaacs, F. J., Jewett, M. C. 2025

  Abstract

  Genetic code expansion with non-canonical amino acids (ncAAs) opens new opportunities for the function and design of proteins by broadening their chemical repertoire. Unfortunately, ncAA incorporation is limited both by a small collection of orthogonal aminoacyl-tRNA synthetases (aaRSs) and tRNAs and by low-throughput methods to discover them. Here, we report the discovery, characterization, and engineering of a UGA suppressing orthogonal translation system mined from metagenomic data. We developed an integrated computational and experimental pipeline to profile the orthogonality of >200 tRNAs, test >1,250 combinations of aaRS:tRNA pairs, and identify the AP1 TrpRS:tRNATrp UCA as an orthogonal pair that natively encodes tryptophan at the UGA codon. We demonstrate that the AP1 TrpRS:tRNATrp UCA is highly active in cell-free and cellular contexts. We then use Ochre, a genomically recoded Escherichia coli strain that lacks UAG and UGA codons, to engineer an AP1 TrpRS variant capable of 5-hydroxytryptophan incorporation at an open UGA codon. We anticipate that our strategy of integrating metagenomic bioprospecting with cell-free screening and cell-based engineering will accelerate the discovery and optimization of orthogonal translation systems for genetic code expansion.

  View details for DOI 10.1101/2025.10.30.685624

  View details for PubMedID 41279103

  View details for PubMedCentralID PMC12636597

• Characterizing and engineering post-translational modifications with high-throughput cell-free expression. Nature communications Wong, D. A., Shaver, Z. M., Cabezas, M. D., Daniel-Ivad, M., Warfel, K. F., Prasanna, D. V., Sobol, S. E., Fernandez, R., Tobias, F., Filip, S. K., Hulbert, S. W., Faull, P., Nicol, R., DeLisa, M. P., Balskus, E. P., Karim, A. S., Jewett, M. C. 2025; 16 (1): 7215

  Abstract

  Post-translational modifications (PTMs) are important for the stability and function of many therapeutic proteins and peptides. Current methods for studying and engineering PTMs are often limited by low-throughput experimental techniques. Here we describe a generalizable, in vitro workflow coupling cell-free gene expression (CFE) with AlphaLISA for the rapid expression and testing of PTM installing proteins. We apply our workflow to two representative classes of peptide and protein therapeutics: ribosomally synthesized and post-translationally modified peptides (RiPPs) and glycoproteins. First, we demonstrate how our workflow can be used to characterize the binding activity of RiPP recognition elements, an important first step in RiPP biosynthesis, and be integrated into a biodiscovery pipeline for computationally predicted RiPP products. Then, we adapt our workflow to study and engineer oligosaccharyltransferases (OSTs) involved in protein glycan coupling technology, leading to the identification of mutant OSTs and sites within a model vaccine carrier protein that enable high efficiency production of glycosylated proteins. We expect that our workflow will accelerate design-build-test-learn cycles for engineering PTMs.

  View details for DOI 10.1038/s41467-025-60526-6

  View details for PubMedID 40764296

  View details for PubMedCentralID 8844085

• Carbon-negative production of acetone and isopropanol by gas fermentation at industrial pilot scale (vol 40, pg 335, 2022) NATURE BIOTECHNOLOGY Liew, F., Nogle, R., Abdalla, T., Rasor, B. J., Canter, C., Jensen, R. O., Wang, L., Strutz, J., Chirania, P., De Tissera, S., Mueller, A. P., Ruan, Z., Gao, A., Tran, L., Engle, N. L., Bromley, J. C., Daniell, J., Conrado, R., Tschaplinski, T. J., Giannone, R. J., Hettich, R. L., Karim, A. S., Simpson, S. D., Brown, S. D., Leang, C., Jewett, M. C., Koepke, M. 2025

  View details for DOI 10.1038/s41587-025-02767-w

  View details for Web of Science ID 001533046400001

  View details for PubMedID 40691324

• Discovery of a single-subunit oligosaccharyltransferase that enables glycosylation of full-length IgG antibodies in bacteria. Nature communications Sotomayor, B., Donahue, T. C., Mahajan, S. P., Taw, M. N., Hulbert, S. W., Bidstrup, E. J., Owitipana, D. N., Pang, A., Yang, X., Ghosal, S., Alabi, C. A., Azadi, P., Gray, J. J., Jewett, M. C., Wang, L. X., DeLisa, M. P. 2025; 16 (1): 6152

  Abstract

  Human immunoglobulin G (IgG) antibodies are a major class of biotherapeutics and undergo N-linked glycosylation in their Fc domain, which is critical for immune functions and therapeutic activity. Hence, technologies for producing authentically glycosylated IgGs are in high demand. Previous attempts to engineer Escherichia coli for this purpose have met limited success due in part to the lack of oligosaccharyltransferase (OST) enzymes that can install N-glycans at the conserved N297 site in the Fc region. Here, we identify a single-subunit OST from Desulfovibrio marinus with relaxed substrate specificity that catalyzes glycosylation of native Fc acceptor sites. By chemoenzymatic remodeling the attached bacterial glycans to homogeneous, asialo complex-type G2 N-glycans, the E. coli-derived Fc binds human FcγRIIIa/CD16a, a key receptor for antibody-dependent cellular cytotoxicity (ADCC). Overall, the discovery of D. marinus OST provides previously unavailable biocatalytic capabilities and sets the stage for using E. coli to produce fully human antibodies.

  View details for DOI 10.1038/s41467-025-61440-7

  View details for PubMedID 40610439

  View details for PubMedCentralID 3201773

• Can protein expression be 'solved'? Trends in biotechnology Baranowski, C., Martin, H. G., Oyarzún, D. A., Spinner, A., Desai, B., Petzold, C. J., Nikolados, E. M., Jaaks-Kraatz, S., Gaber, A., Chalkley, R. J., Scannell, D., Sevey, R., Jewett, M. C., Kelly, P. J., DeBenedictis, E. A. 2025

  Abstract

  Recombinant protein expression is central to biotechnology's application. However, not all proteins can be expressed in all organisms, and, given the vast experimental space, it can be challenging to identify the conditions that will yield successful protein expression. The field lacks a predictive model of soluble protein expression that could replace laborious experimental trial and error. Here, we discuss the state of the field and identify the lack of large, high-fidelity datasets as the primary bottleneck to progress. We outline a proposed path toward an extensible experimental platform for collecting soluble overexpression data across organisms. We suggest that the resulting data should be used to train predictive models of protein expression toward answering the question: can protein expression be solved?

  View details for DOI 10.1016/j.tibtech.2025.04.021

  View details for PubMedID 40461315

• Glycosylation of Structured Protein Domains in Cell-Free Reaction Environments. ACS synthetic biology Bidstrup, E. J., Hill, K., Bandi, C. K., Owitipana, D. N., Chisti, A., Aw, R., Yang, X., Azadi, P., Jewett, M. C., Wang, L. X., Kightlinger, W., DeLisa, M. P. 2025

  Abstract

  The production of N-linked glycoproteins in genetically tractable bacterial hosts and their cell-free extracts holds great promise for low-cost, customizable, and distributed biomanufacturing of glycoconjugate vaccines and glycoprotein therapeutics. In nearly all bacterial N-linked protein glycosylation systems described so far, a single-subunit, transmembrane oligosaccharyltransferase (OST) is employed which favors acceptor sites in flexible, solvent-exposed motifs of the glycoprotein substrate. Yet despite this preference, acceptor sites in structured domains can also be glycosylated in living bacteria, presumably by a mechanism where the site is presented to the OST in a flexible form during or after the membrane translocation step but prior to folding being completed. While N-glycoprotein biosynthesis can also be accomplished using cell-free extracts derived from glycosylation-competent bacteria, it remains to be determined whether the cell-free reaction environment involves a similar mechanism for glycosylation of structured domains. Using anEscherichia coli-based cell-free glycoprotein synthesis (CFGpS) system, we observed efficient glycosylation of two eukaryotic glycoproteins, namely ribonuclease A (RNase A) and the fragment crystallizable (Fc) region of human immunoglobulin G (IgG), whose acceptor sites occur in structurally constrained regions that were not glycosylated when the proteins were already folded. Because this cell-free glycosylation depended on ribosomal translation but not on signal peptide-mediated translocation, we propose the existence of a unique cotranslational, but not cotranslocational, glycosylation mechanism in CFGpS. Collectively, these findings reveal the potential for CFGpS to become a viable platform for producing complex eukaryotic glycoprotein targets.

  View details for DOI 10.1021/acssynbio.5c00229

  View details for PubMedID 40437661

• Scalable Cell-Free Production of Active T7 RNA Polymerase. Biotechnology and bioengineering Rezvani, R. N., Aw, R., Chan, W., Satish, K., Chen, H., Lavy, A., Rimal, S., Patel, D. A., Rao, G., Swartz, J. R., DeLisa, M. P., Kvam, E., Karim, A. S., Krüger, A., Kightlinger, W., Jewett, M. C. 2025

  Abstract

  The SARS-CoV-2 pandemic highlighted the urgent need for biomanufacturing paradigms that are robust and fast. Here, we demonstrate the rapid process development and scalable cell-free production of T7 RNA polymerase, a critical component in mRNA vaccine synthesis. We carry out a 1-L cell-free gene expression (CFE) reaction that achieves over 90% purity, low endotoxin levels, and enhanced activity relative to commercial T7 RNA polymerase. To achieve this demonstration, we implement rolling circle amplification to circumvent difficulties in DNA template generation, and tune cell-free reaction conditions, such as temperature, additives, purification tags, and agitation, to boost yields. We achieve production of a similar quality and titer of T7 RNA polymerase over more than four orders of magnitude in reaction volume. This proof of principle positions CFE as a viable solution for decentralized biotherapeutic manufacturing, enhancing preparedness for future public health crises or emergent threats.

  View details for DOI 10.1002/bit.28993

  View details for PubMedID 40296704

• Semiautomated Production of Cell-Free Biosensors. ACS synthetic biology Brown, D. M., Phillips, D. A., Garcia, D. C., Arce, A., Lucci, T., Davies, J. P., Mangini, J. T., Rhea, K. A., Bernhards, C. B., Biondo, J. R., Blum, S. M., Cole, S. D., Lee, J. A., Lee, M. S., McDonald, N. D., Wang, B., Perdue, D. L., Bower, X. S., Thavarajah, W., Karim, A. S., Lux, M. W., Jewett, M. C., Miklos, A. E., Lucks, J. B. 2025

  Abstract

  Cell-free synthetic biology biosensors have potential as effective in vitro diagnostic technologies for the detection of chemical compounds, such as toxins and human health biomarkers. They have several advantages over conventional laboratory-based diagnostic approaches, including the ability to be assembled, freeze-dried, distributed, and then used at the point of need. This makes them an attractive platform for cheap and rapid chemical detection across the globe. Though promising, a major challenge is scaling up biosensor manufacturing to meet the needs of their multiple uses. Currently, cell-free biosensor assembly during lab-scale development is mostly performed manually by the operator, leading to quality control and performance variability issues. Here we explore the use of liquid-handling robotics to manufacture cell-free biosensor reactions. We compare both manual and semiautomated reaction assembly approaches using the Opentrons OT-2 liquid handling platform on two different cell-free gene expression assay systems that constitutively produce colorimetric (LacZ) or fluorescent (GFP) signals. We test the designed protocol by constructing an entire 384-well plate of fluoride-sensing cell-free biosensors and demonstrate that they perform close to expected detection outcomes.

  View details for DOI 10.1021/acssynbio.4c00703

  View details for PubMedID 40073441

• Cell-Free Expression of Soluble Leafhopper Proteins from Brochosomes. ACS synthetic biology Lay, C. G., Burks, G. R., Li, Z., Barrick, J. E., Schroeder, C. M., Karim, A. S., Jewett, M. C. 2025

  Abstract

  Brochosomes are proteinaceous nanostructures produced by leafhopper insects with superhydrophobic and antireflective properties. Unfortunately, the production and study of brochosome-based materials has been limited by poor understanding of their major constituent subunit proteins, known as brochosomins, as well as their sensitivity to redox conditions due to essential disulfide bonds. Here, we used cell-free gene expression (CFE) to achieve recombinant production and analysis of brochosomin proteins. Through the optimization of redox environment, reaction temperature, and disulfide bond isomerase concentration, we achieved soluble brochosomin yields of up to 341 ± 30 mug/mL. Analysis using dynamic light scattering and transmission electron microscopy revealed distinct aggregation patterns among cell-free mixtures with different expressed brochosomins. We anticipate that the CFE methods developed here will accelerate the ability to change the geometries and properties of natural and modified brochosomes, as well as facilitate the expression and structural analysis of other poorly understood protein complexes.

  View details for DOI 10.1021/acssynbio.4c00773

  View details for PubMedID 40052868

• Exploring the potential landscape of chemical engineering science NATURE CHEMICAL ENGINEERING Adjiman, C. S., Angeli, P., Bardow, A., Bent, S. F., Brandon, N., Galloway, K., Gorte, R. J., Guillen-Gosalbez, G., Gutierrez-Antonio, C., Hatzell, M. C., Jewett, M. C., Kanga, M., Kopke, M., Kraft, M., Lee, U., Liu, Y., Ma, G., Marek, E., Morbidelli, M., Nikolla, E., Papathanasiou, M., Park, A., Pinnau, I., Qiao, S., Ranade, V. V., Ricardez-Sandoval, L., Rivera-Jimenez, S. M., Sahu, K., Smit, B., Snurr, R. Q., Soares, C., Solomon, K., Takanabe, K., Wang, X., Wei, F., Wessling, M., Whitehead, K., Woodley, J. M., Xie, Z., Yan, Y. 2025; 2 (1): 19-25

  View details for DOI 10.1038/s44286-025-00172-3

  View details for Web of Science ID 001550567200019

• Cell-Free Gene Expression: Methods and Applications. Chemical reviews Hunt, A. C., Rasor, B. J., Seki, K., Ekas, H. M., Warfel, K. F., Karim, A. S., Jewett, M. C. 2024

  Abstract

  Cell-free gene expression (CFE) systems empower synthetic biologists to build biological molecules and processes outside of living intact cells. The foundational principle is that precise, complex biomolecular transformations can be conducted in purified enzyme or crude cell lysate systems. This concept circumvents mechanisms that have evolved to facilitate species survival, bypasses limitations on molecular transport across the cell wall, and provides a significant departure from traditional, cell-based processes that rely on microscopic cellular "reactors." In addition, cell-free systems are inherently distributable through freeze-drying, which allows simple distribution before rehydration at the point-of-use. Furthermore, as cell-free systems are nonliving, they provide built-in safeguards for biocontainment without the constraints attendant on genetically modified organisms. These features have led to a significant increase in the development and use of CFE systems over the past two decades. Here, we discuss recent advances in CFE systems and highlight how they are transforming efforts to build cells, control genetic networks, and manufacture biobased products.

  View details for DOI 10.1021/acs.chemrev.4c00116

  View details for PubMedID 39700225

• Developing, Characterizing, and Modeling CRISPR-Based Point-of-Use Pathogen Diagnostics. ACS synthetic biology Jung, J. K., Dreyer, K. S., Dray, K. E., Muldoon, J. J., George, J., Shirman, S., Cabezas, M. D., d'Aquino, A. E., Verosloff, M. S., Seki, K., Rybnicky, G. A., Alam, K. K., Bagheri, N., Jewett, M. C., Leonard, J. N., Mangan, N. M., Lucks, J. B. 2024

  Abstract

  Recent years have seen intense interest in the development of point-of-care nucleic acid diagnostic technologies to address the scaling limitations of laboratory-based approaches. Chief among these are combinations of isothermal amplification approaches with CRISPR-based detection and readouts of target products. Here, we contribute to the growing body of rapid, programmable point-of-care pathogen tests by developing and optimizing a one-pot NASBA-Cas13a nucleic acid detection assay. This test uses the isothermal amplification technique NASBA to amplify target viral nucleic acids, followed by the Cas13a-based detection of amplified sequences. We first demonstrate an in-house formulation of NASBA that enables the optimization of individual NASBA components. We then present design rules for NASBA primer sets and LbuCas13a guide RNAs for the fast and sensitive detection of SARS-CoV-2 viral RNA fragments, resulting in 20-200 aM sensitivity. Finally, we explore the combination of high-throughput assay condition screening with mechanistic ordinary differential equation modeling of the reaction scheme to gain a deeper understanding of the NASBA-Cas13a system. This work presents a framework for developing a mechanistic understanding of reaction performance and optimization that uses both experiments and modeling, which we anticipate will be useful in developing future nucleic acid detection technologies.

  View details for DOI 10.1021/acssynbio.4c00469

  View details for PubMedID 39670656

• Discovery of a single-subunit oligosaccharyltransferase that enables glycosylation of full-length IgG antibodies in Escherichia coli. bioRxiv : the preprint server for biology Sotomayor, B., Donahue, T. C., Mahajan, S. P., Taw, M. N., Hulbert, S. W., Bidstrup, E. J., Owitipana, D. N., Pang, A., Yang, X., Ghosal, S., Alabi, C. A., Azadi, P., Gray, J. J., Jewett, M. C., Wang, L. X., DeLisa, M. P. 2024

  Abstract

  Human immunoglobulin G (IgG) antibodies are one of the most important classes of biotherapeutic agents and undergo glycosylation at the conserved N297 site in the CH2 domain, which is critical for IgG Fc effector functions and anti-inflammatory activity. Hence, technologies for producing authentically glycosylated IgGs are in high demand. While attempts to engineer Escherichia coli for this purpose have been described, they have met limited success due in part to the lack of available oligosaccharyltransferase (OST) enzymes that can install N-linked glycans within the QYNST sequon of the IgG CH2 domain. Here, we identified a previously uncharacterized single-subunit OST (ssOST) from the bacterium Desulfovibrio marinus that exhibited greatly relaxed substrate specificity and, as a result, was able to catalyze glycosylation of native CH2 domains in the context of both a hinge-Fc fragment and a full-length IgG. Although the attached glycans were bacterial in origin, conversion to a homogeneous, asialo complex-type G2 N-glycan at the QYNST sequon of the E. coli-derived hinge-Fc was achieved via chemoenzymatic glycan remodeling. Importantly, the resulting G2-hinge-Fc exhibited strong binding to human FcγRIIIa (CD16a), one of the most potent receptors for eliciting antibody-dependent cellular cytotoxicity (ADCC). Taken together, the discovery of a unique ssOST from D. marinus provides previously unavailable biocatalytic capabilities to the bacterial glycoprotein engineering toolbox and opens the door to using E. coli for the production and glycoengineering of human IgGs and fragments derived thereof.

  View details for DOI 10.1101/2024.08.12.607630

  View details for PubMedID 39574765

  View details for PubMedCentralID PMC11580905

• An Automated Cell-Free Workflow for Transcription Factor Engineering. ACS synthetic biology Ekas, H. M., Wang, B., Silverman, A. D., Lucks, J. B., Karim, A. S., Jewett, M. C. 2024

  Abstract

  The design and optimization of metabolic pathways, genetic systems, and engineered proteins rely on high-throughput assays to streamline design-build-test-learn cycles. However, assay development is a time-consuming and laborious process. Here, we create a generalizable approach for the tailored optimization of automated cell-free gene expression (CFE)-based workflows, which offers distinct advantages over in vivo assays in reaction flexibility, control, and time to data. Centered around designing highly accurate and precise transfers on the Echo Acoustic Liquid Handler, we introduce pilot assays and validation strategies for each stage of protocol development. We then demonstrate the efficacy of our platform by engineering transcription factor-based biosensors. As a model, we rapidly generate and assay libraries of 127 MerR and 134 CadR transcription factor variants in 3682 unique CFE reactions in less than 48 h to improve limit of detection, selectivity, and dynamic range for mercury and cadmium detection. This was achieved by assessing a panel of ligand conditions for sensitivity (to 0.1, 1, 10 muM Hg and 0, 1, 10, 100 muM Cd for MerR and CadR, respectively) and selectivity (against Ag, As, Cd, Co, Cu, Hg, Ni, Pb, and Zn). We anticipate that our Echo-based, cell-free approach can be used to accelerate multiple design workflows in synthetic biology.

  View details for DOI 10.1021/acssynbio.4c00471

  View details for PubMedID 39373325

• What is chemical biology? CELL CHEMICAL BIOLOGY Antolin, A. A., Aye, Y., Bar-Peled, L., De Vita, E., Dudkina, N., Jewett, M. C., Kiely-Collins, H., Mazitschek, R., Zhang, Z. 2024; 31 (9): 1562-1565

  Abstract

  Since its inception, the chemical biology field has undergone significant evolution, with its definition varying greatly based on individual perspectives. For the September 30th anniversary special issue of Cell Chemical Biology, we asked our readers from a range of backgrounds, what is chemical biology?

  View details for Web of Science ID 001348005300001

  View details for PubMedID 39303695

• Engineering a PbrR-Based Biosensor for Cell-Free Detection of Lead at the Legal Limit. ACS synthetic biology Ekas, H. M., Wang, B., Silverman, A. D., Lucks, J. B., Karim, A. S., Jewett, M. C. 2024

  Abstract

  Industrialization and failing infrastructure have led to a growing number of irreversible health conditions resulting from chronic lead exposure. While state-of-the-art analytical chemistry methods provide accurate and sensitive detection of lead, they are too slow, expensive, and centralized to be accessible to many. Cell-free biosensors based on allosteric transcription factors (aTFs) can address the need for accessible, on-demand lead detection at the point of use. However, known aTFs, such as PbrR, are unable to detect lead at concentrations regulated by the Environmental Protection Agency (24-72 nM). Here, we develop a rapid cell-free platform for engineering aTF biosensors with improved sensitivity, selectivity, and dynamic range characteristics. We apply this platform to engineer PbrR mutants for a shift in limit of detection from 10 μM to 50 nM lead and demonstrate use of PbrR as a cell-free biosensor. We envision that our workflow could be applied to engineer any aTF.

  View details for DOI 10.1021/acssynbio.4c00456

  View details for PubMedID 39255329

• Alternate conformational trajectories in ribosome translocation. PLoS computational biology Alejo, J. L., Girodat, D., Hammerling, M. J., Willi, J. A., Jewett, M. C., Engelhart, A. E., Adamala, K. P. 2024; 20 (8): e1012319

  Abstract

  Translocation in protein synthesis entails the efficient and accurate movement of the mRNA-[tRNA]2 substrate through the ribosome after peptide bond formation. An essential conformational change during this process is the swiveling of the small subunit head domain about two rRNA 'hinge' elements. Using iterative selection and molecular dynamics simulations, we derive alternate hinge elements capable of translocation in vitro and in vivo and describe their effects on the conformational trajectory of the EF-G-bound, translocating ribosome. In these alternate conformational pathways, we observe a diversity of swivel kinetics, hinge motions, three-dimensional head domain trajectories and tRNA dynamics. By finding alternate conformational pathways of translocation, we identify motions and intermediates that are essential or malleable in this process. These findings highlight the plasticity of protein synthesis and provide a more thorough understanding of the available sequence and conformational landscape of a central biological process.

  View details for DOI 10.1371/journal.pcbi.1012319

  View details for PubMedID 39141679

• A synthetic cell-free pathway for biocatalytic upgrading of one-carbon substrates. bioRxiv : the preprint server for biology Landwehr, G. M., Vogeli, B., Tian, C., Singal, B., Gupta, A., Lion, R., Sargent, E. H., Karim, A. S., Jewett, M. C. 2024

  Abstract

  Biotechnological processes hold tremendous potential for the efficient and sustainable conversion of one-carbon (C1) substrates into complex multi-carbon products. However, the development of robust and versatile biocatalytic systems for this purpose remains a significant challenge. In this study, we report a hybrid electrochemical-biochemical cell-free system for the conversion of C1 substrates into the universal biological building block acetyl-CoA. The synthetic reductive formate pathway (ReForm) consists of five core enzymes catalyzing non-natural reactions that were established through a cell-free enzyme engineering platform. We demonstrate that ReForm works in a plug-and-play manner to accept diverse C1 substrates including CO2 equivalents. We anticipate that ReForm will facilitate efforts to build and improve synthetic C1 utilization pathways for a formate-based bioeconomy.

  View details for DOI 10.1101/2024.08.08.607227

  View details for PubMedID 39149402

• A frugal CRISPR kit for equitable and accessible education in gene editing and synthetic biology. Nature communications Collins, M., Lau, M. B., Ma, W., Shen, A., Wang, B., Cai, S., La Russa, M., Jewett, M. C., Qi, L. S. 2024; 15 (1): 6563

  Abstract

  Equitable and accessible education in life sciences, bioengineering, and synthetic biology is crucial for training the next generation of scientists, fostering transparency in public decision-making, and ensuring biotechnology can benefit a wide-ranging population. As a groundbreaking technology for genome engineering, CRISPR has transformed research and therapeutics. However, hands-on exposure to this technology in educational settings remains limited due to the extensive resources required for CRISPR experiments. Here, we develop CRISPRkit, an affordable kit designed for gene editing and regulation in high school education. CRISPRkit eliminates the need for specialized equipment, prioritizes biosafety, and utilizes cost-effective reagents. By integrating CRISPRi gene regulation, colorful chromoproteins, cell-free transcription-translation systems, smartphone-based quantification, and an in-house automated algorithm (CRISPectra), our kit offers an inexpensive (~$2) and user-friendly approach to performing and analyzing CRISPR experiments, without the need for a traditional laboratory setup. Experiments conducted by high school students in classroom settings highlight the kit's utility for reliable CRISPRkit experiments. Furthermore, CRISPRkit provides a modular and expandable platform for genome engineering, and we demonstrate its applications for controlling fluorescent proteins and metabolic pathways such as melanin production. We envision CRISPRkit will facilitate biotechnology education for communities of diverse socioeconomic and geographic backgrounds.

  View details for DOI 10.1038/s41467-024-50767-2

  View details for PubMedID 39095367

• Chloroplast Cell-Free Systems from Different Plant Species as a Rapid Prototyping Platform ACS SYNTHETIC BIOLOGY Bohm, C. V., Inckemann, R., Burgis, M., Baumann, J., Brinkmann, C. K., Lipinska, K. E., Gilles, S., Freudigmann, J., Seiler, V. N., Clark, L. G., Jewett, M. C., Voll, L. M., Niederholtmeyer, H. 2024

  Abstract

  Climate change poses a significant threat to global agriculture, necessitating innovative solutions. Plant synthetic biology, particularly chloroplast engineering, holds promise as a viable approach to this challenge. Chloroplasts present a variety of advantageous traits for genetic engineering, but the development of genetic tools and genetic part characterization in these organelles is hindered by the lengthy time scales required to generate transplastomic organisms. To address these challenges, we have established a versatile protocol for generating highly active chloroplast-based cell-free gene expression (CFE) systems derived from a diverse range of plant species, including wheat (monocot), spinach, and poplar trees (dicots). We show that these systems work with conventionally used T7 RNA polymerase as well as the endogenous chloroplast polymerases, allowing for detailed characterization and prototyping of regulatory sequences at both transcription and translation levels. To demonstrate the platform for characterization of promoters and 5' and 3' untranslated regions (UTRs) in higher plant chloroplast gene expression, we analyze a collection of 23 5'UTRs, 10 3'UTRs, and 6 chloroplast promoters, assessed their expression in spinach and wheat extracts, and found consistency in expression patterns, suggesting cross-species compatibility. Looking forward, our chloroplast CFE systems open new avenues for plant synthetic biology, offering prototyping tools for both understanding gene expression and developing engineered plants, which could help meet the demands of a changing global climate.

  View details for DOI 10.1021/acssynbio.4c00117

  View details for Web of Science ID 001273641200001

  View details for PubMedID 39028299

• Establishing a High-Yield Chloroplast Cell-Free System for Prototyping Genetic Parts. ACS synthetic biology Clark, L., Voigt, C. A., Jewett, M. C. 2024

  Abstract

  Plastid engineering offers the potential to carry multigene traits in plants; however, it requires reliable genetic parts to balance expression. The difficulty of chloroplast transformation and slow plant growth makes it challenging to build plants just to characterize genetic parts. To address these limitations, we developed a high-yield cell-free system from Nicotiana tabacum chloroplast extracts for prototyping genetic parts. Our cell-free system uses combined transcription and translation driven by T7 RNA polymerase and works with plasmid or linear template DNA. To develop our system, we optimized lysis, extract preparation procedures (e.g., runoff reaction, centrifugation, and dialysis), and the physiochemical reaction conditions. Our cell-free system can synthesize 34 ± 1 mug/mL luciferase in batch reactions and 60 ± 4 mug/mL in semicontinuous reactions. We apply our batch reaction system to test a library of 103 ribosome binding site (RBS) variants and rank them based on cell-free gene expression. We observe a 1300-fold dynamic range of luciferase expression when normalized by maximum mRNA expression, as assessed by the malachite green aptamer. We also find that the observed normalized gene expression in chloroplast extracts and the predictions made by the RBS Calculator are correlated. We anticipate that chloroplast cell-free systems will increase the speed and reliability of building genetic systems in plant chloroplasts for diverse applications.

  View details for DOI 10.1021/acssynbio.4c00111

  View details for PubMedID 39023433

• Bacterial glycoengineering: Cell-based and cell-free routes for producing biopharmaceuticals with customized glycosylation. Current opinion in chemical biology Palma, J. A., Bunyatov, M. I., Hulbert, S. W., Jewett, M. C., DeLisa, M. P. 2024; 81: 102500

  Abstract

  Glycosylation plays a pivotal role in tuning the folding and function of proteins. Because most human therapeutic proteins are glycosylated, understanding and controlling glycosylation is important for the design, optimization, and manufacture of biopharmaceuticals. Unfortunately, natural eukaryotic glycosylation pathways are complex and often produce heterogeneous glycan patterns, making the production of glycoproteins with chemically precise and homogeneous glycan structures difficult. To overcome these limitations, bacterial glycoengineering has emerged as a simple, cost-effective, and scalable approach to produce designer glycoprotein therapeutics and vaccines in which the glycan structures are engineered to reduce heterogeneity and improve biological and biophysical attributes of the protein. Here, we discuss recent advances in bacterial cell-based and cell-free glycoengineering that have enabled the production of biopharmaceutical glycoproteins with customized glycan structures.

  View details for DOI 10.1016/j.cbpa.2024.102500

  View details for PubMedID 38991462

• Cell-Free Translation Quantification via a Fluorescent Minihelix. ACS synthetic biology Willi, J. A., Karim, A. S., Jewett, M. C. 2024

  Abstract

  Cell-free gene expression systems are used in numerous applications, including medicine making, diagnostics, and educational kits. Accurate quantification of nonfluorescent proteins in these systems remains a challenge. To address this challenge, we report the adaptation and use of an optimized tetra-cysteine minihelix both as a fusion protein and as a standalone reporter with the FlAsH dye. The fluorescent reporter helix is short enough to be encoded on a primer pair to tag any protein of interest via PCR. Both the tagged protein and the standalone reporter can be detected quantitatively in real time or at the end of cell-free expression reactions with standard 96/384-well plate readers, an RT-qPCR system, or gel electrophoresis without the need for staining. The fluorescent signal is stable and correlates linearly with the protein concentration, enabling product quantification. We modified the reporter to study cell-free expression dynamics and engineered ribosome activity. We anticipate that the fluorescent minihelix reporter will facilitate efforts in engineering in vitro transcription and translation systems.

  View details for DOI 10.1021/acssynbio.4c00266

  View details for PubMedID 38979618

• Developing, characterizing and modeling CRISPR-based point-of-use pathogen diagnostics. bioRxiv : the preprint server for biology Jung, J. K., Dreyer, K. S., Dray, K. E., Muldoon, J. J., George, J., Shirman, S., Cabezas, M. D., D'Aquino, A. E., Verosloff, M. S., Seki, K., Rybnicky, G. A., Alam, K. K., Bagheri, N., Jewett, M. C., Leonard, J. N., Mangan, N. M., Lucks, J. B. 2024

  Abstract

  Recent years have seen intense interest in the development of point-of-care nucleic acid diagnostic technologies to address the scaling limitations of laboratory-based approaches. Chief among these are combinations of isothermal amplification approaches with CRISPR-based detection and readouts of target products. Here, we contribute to the growing body of rapid, programmable point-of-care pathogen tests by developing and optimizing a one-pot NASBA-Cas13a nucleic acid detection assay. This test uses the isothermal amplification technique NASBA to amplify target viral nucleic acids, followed by Cas13a-based detection of amplified sequences. We first demonstrate an in-house formulation of NASBA that enables optimization of individual NASBA components. We then present design rules for NASBA primer sets and LbuCas13a guide RNAs for fast and sensitive detection of SARS-CoV-2 viral RNA fragments, resulting in 20 - 200 aM sensitivity without any specialized equipment. Finally, we explore the combination of high-throughput assay condition screening with mechanistic ordinary differential equation modeling of the reaction scheme to gain a deeper understanding of the NASBA-Cas13a system. This work presents a framework for developing a mechanistic understanding of reaction performance and optimization that uses both experiments and modeling, which we anticipate will be useful in developing future nucleic acid detection technologies.

  View details for DOI 10.1101/2024.07.03.601853

  View details for PubMedID 39005318

  View details for PubMedCentralID PMC11244977

• Establishing a Cell-Free Glycoprotein Synthesis System for Enzymatic N-GlcNAcylation. ACS chemical biology DeWinter, M. A., Wong, D. A., Fernandez, R., Kightlinger, W., Thames, A. H., DeLisa, M. P., Jewett, M. C. 2024

  Abstract

  N-linked glycosylation plays a key role in the efficacy of many therapeutic proteins. One limitation to the bacterial glycoengineering of human N-linked glycans is the difficulty of installing a single N-acetylglucosamine (GlcNAc), the reducing end sugar of many human-type glycans, onto asparagine in a single step (N-GlcNAcylation). Here, we develop an in vitro method for N-GlcNAcylating proteins using the oligosaccharyltransferase PglB from Campylobacter jejuni. We use cell-free protein synthesis (CFPS) to test promiscuous PglB variants previously reported in the literature for the ability to produce N-GlcNAc and successfully determine that PglB with an N311V mutation (PglBN311V) exhibits increased GlcNAc transferase activity relative to the wild-type enzyme. We then improve the transfer efficiency by producing CFPS extracts enriched with PglBN311V and further optimize the reaction conditions, achieving a 98.6 ± 0.5% glycosylation efficiency. We anticipate this method will expand the glycoengineering toolbox for therapeutic research and biomanufacturing.

  View details for DOI 10.1021/acschembio.4c00228

  View details for PubMedID 38934647

• Deconstructing synthetic biology across scales: a conceptual approach for training synthetic biologists. Nature communications Karim, A. S., Brown, D. M., Archuleta, C. M., Grannan, S., Aristilde, L., Goyal, Y., Leonard, J. N., Mangan, N. M., Prindle, A., Rocklin, G. J., Tyo, K. J., Zoloth, L., Jewett, M. C., Calkins, S., Kamat, N. P., Tullman-Ercek, D., Lucks, J. B. 2024; 15 (1): 5425

  Abstract

  Synthetic biology allows us to reuse, repurpose, and reconfigure biological systems to address society's most pressing challenges. Developing biotechnologies in this way requires integrating concepts across disciplines, posing challenges to educating students with diverse expertise. We created a framework for synthetic biology training that deconstructs biotechnologies across scales-molecular, circuit/network, cell/cell-free systems, biological communities, and societal-giving students a holistic toolkit to integrate cross-disciplinary concepts towards responsible innovation of successful biotechnologies. We present this framework, lessons learned, and inclusive teaching materials to allow its adaption to train the next generation of synthetic biologists.

  View details for DOI 10.1038/s41467-024-49626-x

  View details for PubMedID 38926339

  View details for PubMedCentralID PMC11208543

• Cell-free biosynthesis and engineering of ribosomally synthesized lanthipeptides. Nature communications Liu, W. Q., Ji, X., Ba, F., Zhang, Y., Xu, H., Huang, S., Zheng, X., Liu, Y., Ling, S., Jewett, M. C., Li, J. 2024; 15 (1): 4336

  Abstract

  Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural products with diverse chemical structures and potent biological activities. A vast majority of RiPP gene clusters remain unexplored in microbial genomes, which is partially due to the lack of rapid and efficient heterologous expression systems for RiPP characterization and biosynthesis. Here, we report a unified biocatalysis (UniBioCat) system based on cell-free gene expression for rapid biosynthesis and engineering of RiPPs. We demonstrate UniBioCat by reconstituting a full biosynthetic pathway for de novo biosynthesis of salivaricin B, a lanthipeptide RiPP. Next, we delete several protease/peptidase genes from the source strain to enhance the performance of UniBioCat, which then can synthesize and screen salivaricin B variants with enhanced antimicrobial activity. Finally, we show that UniBioCat is generalizable by synthesizing and evaluating the bioactivity of ten uncharacterized lanthipeptides. We expect UniBioCat to accelerate the discovery, characterization, and synthesis of RiPPs.

  View details for DOI 10.1038/s41467-024-48726-y

  View details for PubMedID 38773100

  View details for PubMedCentralID 5474203

• Phylogenomics and genetic analysis of solvent-producing Clostridium species. Scientific data Jensen, R. O., Schulz, F., Roux, S., Klingeman, D. M., Mitchell, W. P., Udwary, D., Moraïs, S., Reynoso, V., Winkler, J., Nagaraju, S., De Tissera, S., Shapiro, N., Ivanova, N., Reddy, T. B., Mizrahi, I., Utturkar, S. M., Bayer, E. A., Woyke, T., Mouncey, N. J., Jewett, M. C., Simpson, S. D., Köpke, M., Jones, D. T., Brown, S. D. 2024; 11 (1): 432

  Abstract

  The genus Clostridium is a large and diverse group within the Bacillota (formerly Firmicutes), whose members can encode useful complex traits such as solvent production, gas-fermentation, and lignocellulose breakdown. We describe 270 genome sequences of solventogenic clostridia from a comprehensive industrial strain collection assembled by Professor David Jones that includes 194 C. beijerinckii, 57 C. saccharobutylicum, 4 C. saccharoperbutylacetonicum, 5 C. butyricum, 7 C. acetobutylicum, and 3 C. tetanomorphum genomes. We report methods, analyses and characterization for phylogeny, key attributes, core biosynthetic genes, secondary metabolites, plasmids, prophage/CRISPR diversity, cellulosomes and quorum sensing for the 6 species. The expanded genomic data described here will facilitate engineering of solvent-producing clostridia as well as non-model microorganisms with innately desirable traits. Sequences could be applied in conventional platform biocatalysts such as yeast or Escherichia coli for enhanced chemical production. Recently, gene sequences from this collection were used to engineer Clostridium autoethanogenum, a gas-fermenting autotrophic acetogen, for continuous acetone or isopropanol production, as well as butanol, butanoic acid, hexanol and hexanoic acid production.

  View details for DOI 10.1038/s41597-024-03210-6

  View details for PubMedID 38693191

  View details for PubMedCentralID 10538166

• Ribosome Pool Engineering Increases Protein Biosynthesis Yields. ACS central science Kofman, C., Willi, J. A., Karim, A. S., Jewett, M. C. 2024; 10 (4): 871-881

  Abstract

  The biosynthetic capability of the bacterial ribosome motivates efforts to understand and harness sequence-optimized versions for synthetic biology. However, functional differences between natively occurring ribosomal RNA (rRNA) operon sequences remain poorly characterized. Here, we use an in vitro ribosome synthesis and translation platform to measure protein production capabilities of ribosomes derived from all unique combinations of 16S and 23S rRNAs from seven distinct Escherichia coli rRNA operon sequences. We observe that polymorphisms that distinguish native E. coli rRNA operons lead to significant functional changes in the resulting ribosomes, ranging from negligible or low gene expression to matching the protein production activity of the standard rRNA operon B sequence. We go on to generate strains expressing single rRNA operons and show that not only do some purified in vivo expressed homogeneous ribosome pools outperform the wild-type, heterogeneous ribosome pool but also that a crude cell lysate made from the strain expressing only operon A ribosomes shows significant yield increases for a panel of medically and industrially relevant proteins. We anticipate that ribosome pool engineering can be applied as a tool to increase yields across many protein biomanufacturing systems, as well as improve basic understanding of ribosome heterogeneity and evolution.

  View details for DOI 10.1021/acscentsci.3c01413

  View details for PubMedID 38680563

  View details for PubMedCentralID PMC11046459

• Building Synthetic Cells─From the Technology Infrastructure to Cellular Entities. ACS synthetic biology Rothschild, L. J., Averesch, N. J., Strychalski, E. A., Moser, F., Glass, J. I., Cruz Perez, R., Yekinni, I. O., Rothschild-Mancinelli, B., Roberts Kingman, G. A., Wu, F., Waeterschoot, J., Ioannou, I. A., Jewett, M. C., Liu, A. P., Noireaux, V., Sorenson, C., Adamala, K. P. 2024

  Abstract

  The de novo construction of a living organism is a compelling vision. Despite the astonishing technologies developed to modify living cells, building a functioning cell "from scratch" has yet to be accomplished. The pursuit of this goal alone has─and will─yield scientific insights affecting fields as diverse as cell biology, biotechnology, medicine, and astrobiology. Multiple approaches have aimed to create biochemical systems manifesting common characteristics of life, such as compartmentalization, metabolism, and replication and the derived features, evolution, responsiveness to stimuli, and directed movement. Significant achievements in synthesizing each of these criteria have been made, individually and in limited combinations. Here, we review these efforts, distinguish different approaches, and highlight bottlenecks in the current research. We look ahead at what work remains to be accomplished and propose a "roadmap" with key milestones to achieve the vision of building cells from molecular parts.

  View details for DOI 10.1021/acssynbio.3c00724

  View details for PubMedID 38530077

• Using High-Throughput Experiments To Screen N-Glycosyltransferases with Altered Specificities. ACS synthetic biology Lin, L., Kightlinger, W., Warfel, K. F., Jewett, M. C., Mrksich, M. 2024

  Abstract

  The important roles that protein glycosylation plays in modulating the activities and efficacies of protein therapeutics have motivated the development of synthetic glycosylation systems in living bacteria and in vitro. A key challenge is the lack of glycosyltransferases that can efficiently and site-specifically glycosylate desired target proteins without the need to alter primary amino acid sequences at the acceptor site. Here, we report an efficient and systematic method to screen a library of glycosyltransferases capable of modifying comprehensive sets of acceptor peptide sequences in parallel. This approach is enabled by cell-free protein synthesis and mass spectrometry of self-assembled monolayers and is used to engineer a recently discovered prokaryotic N-glycosyltransferase (NGT). We screened 26 pools of site-saturated NGT libraries to identify relevant residues that determine polypeptide specificity and then characterized 122 NGT mutants, using 1052 unique peptides and 52,894 unique reaction conditions. We define a panel of 14 NGTs that can modify 93% of all sequences within the canonical X-1-N-X+1-S/T eukaryotic glycosylation sequences as well as another panel for many noncanonical sequences (with 10 of 17 non-S/T amino acids at the X+2 position). We then successfully applied our panel of NGTs to increase the efficiency of glycosylation for three protein therapeutics. Our work promises to significantly expand the substrates amenable to in vitro and bacterial glycoengineering.

  View details for DOI 10.1021/acssynbio.3c00769

  View details for PubMedID 38526141

• Ribosome Pool Engineering Increases Protein Biosynthesis Yields ACS CENTRAL SCIENCE Kofman, C., Willi, J. A., Karim, A. S., Jewett, M. C. 2024

  View details for DOI 10.1021/acscentsci.3c01413

  View details for Web of Science ID 001189145200001

• Validation of Cell-Free Protein Synthesis Aboard the International Space Station. ACS synthetic biology Kocalar, S., Miller, B. M., Huang, A., Gleason, E., Martin, K., Foley, K., Copeland, D. S., Jewett, M. C., Saavedra, E. A., Kraves, S. 2024

  Abstract

  Cell-free protein synthesis (CFPS) is a rapidly maturing in vitro gene expression platform that can be used to transcribe and translate nucleic acids at the point of need, enabling on-demand synthesis of peptide-based vaccines and biotherapeutics as well as the development of diagnostic tests for environmental contaminants and infectious agents. Unlike traditional cell-based systems, CFPS platforms do not require the maintenance of living cells and can be deployed with minimal equipment; therefore, they hold promise for applications in low-resource contexts, including spaceflight. Here, we evaluate the performance of the cell-free platform BioBits aboard the International Space Station by expressing RNA-based aptamers and fluorescent proteins that can serve as biological indicators. We validate two classes of biological sensors that detect either the small-molecule DFHBI or a specific RNA sequence. Upon detection of their respective analytes, both biological sensors produce fluorescent readouts that are visually confirmed using a hand-held fluorescence viewer and imaged for quantitative analysis. Our findings provide insights into the kinetics of cell-free transcription and translation in a microgravity environment and reveal that both biosensors perform robustly in space. Our findings lay the groundwork for portable, low-cost applications ranging from point-of-care health monitoring to on-demand detection of environmental hazards in low-resource communities both on Earth and beyond.

  View details for DOI 10.1021/acssynbio.3c00733

  View details for PubMedID 38442491

• Progress in Engineering Synthetic Cells and Cell-Free Systems. ACS synthetic biology Dogterom, M., Kamat, N. P., Jewett, M. C., Adamala, K. P. 2024

  View details for DOI 10.1021/acssynbio.4c00100

  View details for PubMedID 38430125

• Cell-Free Systems for the Production of Glycoproteins. Methods in molecular biology (Clifton, N.J.) Bidstrup, E. J., Kwon, Y. H., Kim, K., Bandi, C. K., Aw, R., Jewett, M. C., DeLisa, M. P. 2024; 2762: 309-328

  Abstract

  Cell-free protein synthesis (CFPS), whereby cell lysates are used to produce proteins from a genetic template, has matured as an attractive alternative to standard biomanufacturing modalities due to its high volumetric productivity contained within a distributable platform. Initially, cell-free lysates produced from Escherichia coli, which are both simple to produce and cost-effective for the production of a wide variety of proteins, were unable to produce glycosylated proteins as E. coli lacks native glycosylation machinery. With many important therapeutic proteins possessing asparagine-linked glycans that are critical for structure and function, this gap in CFPS production capabilities was addressed with the development of cell-free expression of glycoproteins (glycoCFE), which uses the supplementation of extracted lipid-linked oligosaccharides and purified oligosaccharyltransferases to enable glycoprotein production in the CFPS reaction environment. In this chapter, we highlight the basic methods for the preparation of reagents for glycoCFE and the protocol for expression and glycosylation of a model protein using a more productive, yet simplified, glycoCFE setup. Beyond this initial protocol, we also highlight how this protocol can be extended to a wide range of alternative glycan structures, oligosaccharyltransferases, and acceptor proteins as well as to a one-pot cell-free glycoprotein synthesis reaction.

  View details for DOI 10.1007/978-1-0716-3666-4_19

  View details for PubMedID 38315374

  View details for PubMedCentralID 9735008

• Improving Cell-Free Expression of Model Membrane Proteins by Tuning Ribosome Cotranslational Membrane Association and Nascent Chain Aggregation. ACS synthetic biology Steinkuhler, J., Peruzzi, J. A., Kruger, A., Villasenor, C. G., Jacobs, M. L., Jewett, M. C., Kamat, N. P. 2023

  Abstract

  Cell-free gene expression (CFE) systems are powerful tools for transcribing and translating genes outside of a living cell. Synthesis of membrane proteins is of particular interest, but their yield in CFE is substantially lower than that for soluble proteins. In this paper, we study the CFE of membrane proteins and develop a quantitative kinetic model. We identify that ribosome stalling during the translation of membrane proteins is a strong predictor of membrane protein synthesis due to aggregation between the ribosome nascent chains. Synthesis can be improved by the addition of lipid membranes, which incorporate protein nascent chains and, therefore, kinetically compete with aggregation. We show that the balance between peptide-membrane association and peptide aggregation rates determines the yield of the synthesized membrane protein. We define a membrane protein expression score that can be used to rationalize the engineering of lipid composition and the N-terminal domain of a native and computationally designed membrane proteins produced through CFE.

  View details for DOI 10.1021/acssynbio.3c00357

  View details for PubMedID 38150067

• Enzymatic transfer of a single GlcNAc residue to asparagine in a single-pot in vitro glycosylation reaction DeWinter, M., Wong, D., Kightlinger, W., DeLisa, M., Jewett, M. OXFORD UNIV PRESS INC. 2023: 1073

  View details for Web of Science ID 001182824100256

• Developing a cell-free platform for engineering bacterial oligosaccharyltransferases Wong, D., Warfel, K., Shaver, Z., Sobol, S., Fernandez, R., Jewett, M. OXFORD UNIV PRESS INC. 2023: 1053

  View details for Web of Science ID 001182824100212

• Establishing a versatile toolkit of flux enhanced strains and cell extracts for pathway prototyping. Metabolic engineering Yi, X., Rasor, B. J., Boadi, N., Louie, K., Northen, T. R., Karim, A. S., Jewett, M. C., Alper, H. S. 2023

  Abstract

  Building and optimizing biosynthetic pathways in engineered cells holds promise to address societal needs in energy, materials, and medicine, but it is often time-consuming. Cell-free synthetic biology has emerged as a powerful tool to accelerate design-build-test-learn cycles for pathway engineering with increased tolerance to toxic compounds. However, most cell-free pathway prototyping to date has been performed in extracts from wildtype cells which often do not have sufficient flux towards the pathways of interest, which can be enhanced by engineering. Here, to address this gap, we create a set of engineered Escherichia coli and Saccharomyces cerevisiae strains rewired via CRISPR-dCas9 to achieve high-flux toward key metabolic precursors; namely, acetyl-CoA, shikimate, triose-phosphate, oxaloacetate, α-ketoglutarate, and glucose-6-phosphate. Cell-free extracts generated from these strains are used for targeted enzyme screening in vitro. As model systems, we assess in vivo and in vitro production of triacetic acid lactone from acetyl-CoA and muconic acid from the shikimate pathway. The need for these platforms is exemplified by the fact that muconic acid cannot be detected in wildtype extracts provided with the same biosynthetic enzymes. We also perform metabolomic comparison to understand biochemical differences between the cellular and cell-free muconic acid synthesis systems (E. coli and S. cerevisiae cells and cell extracts with and without metabolic rewiring). While any given pathway has different interfaces with metabolism, we anticipate that this set of pre-optimized, flux enhanced cell extracts will enable prototyping efforts for new biosynthetic pathways and the discovery of biochemical functions of enzymes.

  View details for DOI 10.1016/j.ymben.2023.10.008

  View details for PubMedID 37890611

• At-Home, Cell-Free Synthetic Biology Education Modules for Transcriptional Regulation and Environmental Water Quality Monitoring. ACS synthetic biology Jung, J. K., Rasor, B. J., Rybnicky, G. A., Silverman, A. D., Standeven, J., Kuhn, R., Granito, T., Ekas, H. M., Wang, B. M., Karim, A. S., Lucks, J. B., Jewett, M. C. 2023

  Abstract

  As the field of synthetic biology expands, the need to grow and train science, technology, engineering, and math (STEM) practitioners is essential. However, the lack of access to hands-on demonstrations has led to inequalities of opportunity and practice. In addition, there is a gap in providing content that enables students to make their own bioengineered systems. To address these challenges, we develop four shelf-stable cell-free biosensing educational modules that work by simply adding water and DNA to freeze-dried crude extracts of non-pathogenic Escherichia coli. We introduce activities and supporting curricula to teach the structure and function of the lac operon, dose-responsive behavior, considerations for biosensor outputs, and a "build-your-own" activity for monitoring environmental contaminants in water. We piloted these modules with K-12 teachers and 130 high-school students in their classrooms─and at home─without professional laboratory equipment. This work promises to catalyze access to interactive synthetic biology education opportunities.

  View details for DOI 10.1021/acssynbio.3c00223

  View details for PubMedID 37699423

• A Cell-Free Protein Synthesis Platform to Produce a Clinically Relevant Allergen Panel. ACS synthetic biology Thames, A. H., Rische, C. H., Cao, Y., Krier-Burris, R. A., Kuang, F. L., Hamilton, R. G., Bronzert, C., Bochner, B. S., Jewett, M. C. 2023

  Abstract

  Allergens are used in the clinical diagnosis (e.g., skin tests) and treatment (e.g., immunotherapy) of allergic diseases. With growing interest in molecular allergy diagnostics and precision therapies, new tools are needed for producing allergen-based reagents. As a step to address this need, we demonstrate a cell-free protein synthesis approach for allergen production of a clinically relevant allergen panel composed of common allergens spanning a wide range of phylogenetic kingdoms. We show that allergens produced with this approach can be recognized by allergen-specific immunoglobulin E (IgE), either monoclonals or in patient sera. We also show that a cell-free expressed allergen can activate human cells such as peripheral blood basophils and CD34+ progenitor-derived mast cells in an IgE-dependent manner. We anticipate that this cell-free platform for allergen production will enable diagnostic and therapeutic technologies, providing useful tools and treatments for both the allergist and allergic patient.

  View details for DOI 10.1021/acssynbio.3c00269

  View details for PubMedID 37553068

• Glycovaccinology: The design and engineering of carbohydrate-based vaccine components. Biotechnology advances Hulbert, S. W., Desai, P., Jewett, M. C., DeLisa, M. P., Williams, A. J. 2023: 108234

  Abstract

  Vaccines remain one of the most important pillars in preventative medicine, providing protection against a wide array of diseases by inducing humoral and/or cellular immunity. Of the many possible candidate antigens for subunit vaccine development, carbohydrates are particularly appealing because of their ubiquitous presence on the surface of all living cells, viruses, and parasites as well as their known interactions with both innate and adaptive immune cells. Indeed, several licensed vaccines leverage bacterial cell-surface carbohydrates as antigens for inducing antigen-specific plasma cells secreting protective antibodies and the development of memory T and B cells. Carbohydrates have also garnered attention in other aspects of vaccine development, for example, as adjuvants that enhance the immune response by either activating innate immune responses or targeting specific immune cells. Additionally, carbohydrates can function as immunomodulators that dampen undesired humoral immune responses to entire protein antigens or specific, conserved regions on antigenic proteins. In this review, we highlight how the interplay between carbohydrates and the adaptive and innate arms of the immune response is guiding the development of glycans as vaccine components that act as antigens, adjuvants, and immunomodulators. We also discuss how advances in the field of synthetic glycobiology are enabling the design, engineering, and production of this new generation of carbohydrate-containing vaccine formulations with the potential to prevent infectious diseases, malignancies, and complex immune disorders.

  View details for DOI 10.1016/j.biotechadv.2023.108234

  View details for PubMedID 37558188

• A rapid cell-free expression and screening platform for antibody discovery. Nature communications Hunt, A. C., Vögeli, B., Hassan, A. O., Guerrero, L., Kightlinger, W., Yoesep, D. J., Krüger, A., DeWinter, M., Diamond, M. S., Karim, A. S., Jewett, M. C. 2023; 14 (1): 3897

  Abstract

  Antibody discovery is bottlenecked by the individual expression and evaluation of antigen-specific hits. Here, we address this bottleneck by developing a workflow combining cell-free DNA template generation, cell-free protein synthesis, and binding measurements of antibody fragments in a process that takes hours rather than weeks. We apply this workflow to evaluate 135 previously published antibodies targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including all 8 antibodies previously granted emergency use authorization for coronavirus disease 2019 (COVID-19), and demonstrate identification of the most potent antibodies. We also evaluate 119 anti-SARS-CoV-2 antibodies from a mouse immunized with the SARS-CoV-2 spike protein and identify neutralizing antibody candidates, including the antibody SC2-3, which binds the SARS-CoV-2 spike protein of all tested variants of concern. We expect that our cell-free workflow will accelerate the discovery and characterization of antibodies for future pandemics and for research, diagnostic, and therapeutic applications more broadly.

  View details for DOI 10.1038/s41467-023-38965-w

  View details for PubMedID 37400446

  View details for PubMedCentralID PMC10318062

• Rapid biosynthesis of glycoprotein therapeutics and vaccines from freeze-dried bacterial cell lysates. Nature protocols Stark, J. C., Jaroentomeechai, T., Warfel, K. F., Hershewe, J. M., DeLisa, M. P., Jewett, M. C. 2023

  Abstract

  The advent of distributed biomanufacturing platforms promises to increase agility in biologic production and expand access by reducing reliance on refrigerated supply chains. However, such platforms are not capable of robustly producing glycoproteins, which represent the majority of biologics approved or in development. To address this limitation, we developed cell-free technologies that enable rapid, modular production of glycoprotein therapeutics and vaccines from freeze-dried Escherichia coli cell lysates. Here, we describe a protocol for generation of cell-free lysates and freeze-dried reactions for on-demand synthesis of desired glycoproteins. The protocol includes construction and culture of the bacterial chassis strain, cell-free lysate production, assembly of freeze-dried reactions, cell-free glycoprotein synthesis, and glycoprotein characterization, all of which can be completed in one week or less. We anticipate that cell-free technologies, along with this comprehensive user manual, will help accelerate development and distribution of glycoprotein therapeutics and vaccines.

  View details for DOI 10.1038/s41596-022-00799-z

  View details for PubMedID 37328605

  View details for PubMedCentralID 3567426

• Point-of-Care Peptide Hormone Production Enabled by Cell-Free Protein Synthesis ACS SYNTHETIC BIOLOGY DeWinter, M. A., Thames, A., Guerrero, L., Kightlinger, W., Karim, A. S., Jewett, M. C. 2023; 12 (4): 1216-1226

  Abstract

  In resource-limited settings, it can be difficult to safely deliver sensitive biologic medicines to patients due to cold chain and infrastructure constraints. Point-of-care drug manufacturing could circumvent these challenges since medicines could be produced locally and used on-demand. Toward this vision, we combine cell-free protein synthesis (CFPS) and a 2-in-1 affinity purification and enzymatic cleavage scheme to develop a platform for point-of-care drug manufacturing. As a model, we use this platform to synthesize a panel of peptide hormones, an important class of medications that can be used to treat a wide variety of diseases including diabetes, osteoporosis, and growth disorders. With this approach, temperature-stable lyophilized CFPS reaction components can be rehydrated with DNA encoding a SUMOylated peptide hormone of interest when needed. Strep-Tactin affinity purification and on-bead SUMO protease cleavage yield peptide hormones in their native form that are recognized by ELISA antibodies and that can bind their respective receptors. With further development to ensure proper biologic activity and patient safety, we envision that this platform could be used to manufacture valuable peptide hormone drugs in a decentralized way.

  View details for DOI 10.1021/acssynbio.2c006801216

  View details for Web of Science ID 000975893300001

  View details for PubMedID 36940255

• Community science designed ribosomes with beneficial phenotypes. Nature communications Kruger, A., Watkins, A. M., Wellington-Oguri, R., Romano, J., Kofman, C., DeFoe, A., Kim, Y., Anderson-Lee, J., Fisker, E., Townley, J., Eterna Participants, d'Aquino, A. E., Das, R., Jewett, M. C. 2023; 14 (1): 961

  Abstract

  Functional design of ribosomes with mutant ribosomal RNA (rRNA) can expand opportunities for understanding molecular translation, building cells from the bottom-up, and engineering ribosomes with altered capabilities. However, such efforts are hampered by cell viability constraints, an enormous combinatorial sequence space, and limitations on large-scale, 3D design of RNA structures and functions. To address these challenges, we develop an integrated community science and experimental screening approach for rational design of ribosomes. This approach couples Eterna, an online video game that crowdsources RNA sequence design to community scientists in the form of puzzles, with in vitro ribosome synthesis, assembly, and translation in multiple design-build-test-learn cycles. We apply our framework to discover mutant rRNA sequences that improve protein synthesis in vitro and cell growth in vivo, relative to wild type ribosomes, under diverse environmental conditions. This work provides insights into rRNA sequence-function relationships and has implications for synthetic biology.

  View details for DOI 10.1038/s41467-023-35827-3

  View details for PubMedID 36810740

• Cell-free Biosynthesis of Peptidomimetics BIOTECHNOLOGY AND BIOPROCESS ENGINEERING Lee, K., Willi, J. A., Cho, N., Kim, I., Jewett, M. C., Lee, J. 2023; 28 (6): 905-921

  Abstract

  A wide variety of peptidomimetics (peptide analogs) possessing innovative biological functions have been brought forth as therapeutic candidates through cell-free protein synthesis (CFPS) systems. A key feature of these peptidomimetic drugs is the use of non-canonical amino acid building blocks with diverse biochemical properties that expand functional diversity. Here, we summarize recent technologies leveraging CFPS platforms to expand the reach of peptidomimetics drugs. We also offer perspectives on engineering the translational machinery that may open new opportunities for expanding genetically encoded chemistry to transform drug discovery practice beyond traditional boundaries.

  View details for DOI 10.1007/s12257-022-0268-5

  View details for Web of Science ID 000925804700002

  View details for PubMedID 36778039

  View details for PubMedCentralID PMC9896473

• At-home, cell-free synthetic biology education modules for transcriptional regulation and environmental water quality monitoring. bioRxiv : the preprint server for biology Jung, K. J., Rasor, B. J., Rybnicky, G. A., Silverman, A. D., Standeven, J., Kuhn, R., Granito, T., Ekas, H. M., Wang, B. M., Karim, A. S., Lucks, J. B., Jewett, M. C. 2023

  Abstract

  As the field of synthetic biology expands, the need to grow and train science, technology, engineering, and math (STEM) practitioners is essential. However, the lack of access to hands-on demonstrations has led to inequalities of opportunity and practice. In addition, there is a gap in providing content that enables students to make their own bioengineered systems. To address these challenges, we develop four shelf-stable cell-free biosensing educational modules that work by just-adding-water and DNA to freeze-dried crude extracts of Escherichia coli . We introduce activities and supporting curricula to teach the structure and function of the lac operon, dose-responsive behavior, considerations for biosensor outputs, and a 'build-your-own' activity for monitoring environmental contaminants in water. We piloted these modules with K-12 teachers and 130 high school students in their classrooms - and at home - without professional laboratory equipment or researcher oversight. This work promises to catalyze access to interactive synthetic biology education opportunities.

  View details for DOI 10.1101/2023.01.09.523248

  View details for PubMedID 36711593

  View details for PubMedCentralID PMC9881948

• A low-cost recombinant glycoconjugate vaccine confers immunogenicity and protection against enterotoxigenic Escherichia coli infections in mice. Frontiers in molecular biosciences Williams, A. J., Warfel, K. F., Desai, P., Li, J., Lee, J., Wong, D. A., Nguyen, P. M., Qin, Y., Sobol, S. E., Jewett, M. C., Chang, Y., DeLisa, M. P. 2023; 10: 1085887

  Abstract

  Enterotoxigenic Escherichia coli (ETEC) is the primary etiologic agent of traveler's diarrhea and a major cause of diarrheal disease and death worldwide, especially in infants and young children. Despite significant efforts over the past several decades, an affordable vaccine that appreciably decreases mortality and morbidity associated with ETEC infection among children under the age of 5years remains an unmet aspirational goal. Here, we describe robust, cost-effective biosynthetic routes that leverage glycoengineered strains of non-pathogenic E. coli or their cell-free extracts for producing conjugate vaccine candidates against two of the most prevalent O serogroups of ETEC, O148 and O78. Specifically, we demonstrate site-specific installation of O-antigen polysaccharides (O-PS) corresponding to these serogroups onto licensed carrier proteins using the oligosaccharyltransferase PglB from Campylobacter jejuni. The resulting conjugates stimulate strong O-PS-specific humoral responses in mice and elicit IgG antibodies that possess bactericidal activity against the cognate pathogens. We also show that one of the prototype conjugates decorated with serogroup O148 O-PS reduces ETEC colonization in mice, providing evidence of vaccine-induced mucosal protection. We anticipate that our bacterial cell-based and cell-free platforms will enable creation of multivalent formulations with the potential for broad ETEC serogroup protection and increased access through low-cost biomanufacturing.

  View details for DOI 10.3389/fmolb.2023.1085887

  View details for PubMedID 36936989

• Cell-free Macromolecular Synthesis Preface CELL-FREE MACROMOLECULAR SYNTHESIS Lu, Y., Jewett, M. C. edited by Lu, Y., Jewett, M. C. 2023; 185: VII-VIII

  View details for Web of Science ID 001304459400001

• Cell-free Production System Development Preface CELL-FREE PRODUCTION Lu, Y., Jewett, M. C. edited by Lu, Y., Jewett, M. C. 2023; 186: VII-VIII

  View details for Web of Science ID 001304459800001

• Computationally-guided design and selection of high performing ribosomal active site mutants. Nucleic acids research Kofman, C., Watkins, A. M., Kim, D. S., Willi, J. A., Wooldredge, A. C., Karim, A. S., Das, R., Jewett, M. C. 2022

  Abstract

  Understanding how modifications to the ribosome affect function has implications for studying ribosome biogenesis, building minimal cells, and repurposing ribosomes for synthetic biology. However, efforts to design sequence-modified ribosomes have been limited because point mutations in the ribosomal RNA (rRNA), especially in the catalytic active site (peptidyl transferase center; PTC), are often functionally detrimental. Moreover, methods for directed evolution of rRNA are constrained by practical considerations (e.g. library size). Here, to address these limitations, we developed a computational rRNA design approach for screening guided libraries of mutant ribosomes. Our method includes in silico library design and selection using a Rosetta stepwise Monte Carlo method (SWM), library construction and in vitro testing of combined ribosomal assembly and translation activity, and functional characterization in vivo. As a model, we apply our method to making modified ribosomes with mutant PTCs. We engineer ribosomes with as many as 30 mutations in their PTCs, highlighting previously unidentified epistatic interactions, and show that SWM helps identify sequences with beneficial phenotypes as compared to random library sequences. We further demonstrate that some variants improve cell growth in vivo, relative to wild type ribosomes. We anticipate that SWM design and selection may serve as a powerful tool for rRNA engineering.

  View details for DOI 10.1093/nar/gkac1036

  View details for PubMedID 36484094

• Three-dimensional structure-guided evolution of a ribosome with tethered subunits. Nature chemical biology Kim, D. S., Watkins, A., Bidstrup, E., Lee, J., Topkar, V., Kofman, C., Schwarz, K. J., Liu, Y., Pintilie, G., Roney, E., Das, R., Jewett, M. C. 2022

  Abstract

  RNA-based macromolecular machines, such as the ribosome, have functional parts reliant on structural interactions spanning sequence-distant regions. These features limit evolutionary exploration of mutant libraries and confound three-dimensional structure-guided design. To address these challenges, we describe Evolink (evolution and linkage), a method that enables high-throughput evolution of sequence-distant regions in large macromolecular machines, and library design guided by computational RNA modeling to enable exploration of structurally stable designs. Using Evolink, we evolved a tethered ribosome with a 58% increased activity in orthogonal protein translation and a 97% improvement in doubling times in SQ171 cells compared to a previously developed tethered ribosome, and reveal new permissible sequences in a pair of ribosomal helices with previously explored biological function. The Evolink approach may enable enhanced engineering of macromolecular machines for new and improved functions for synthetic biology.

  View details for DOI 10.1038/s41589-022-01064-w

  View details for PubMedID 35836020

• Characterizing and Controlling Nanoscale Self-Assembly of Suckerin-12 ACS SYNTHETIC BIOLOGY Hershewe, J. M., Wiseman, W. D., Kath, J. E., Buck, C. C., Gupta, M. K., Dennis, P. B., Naik, R. R., Jewett, M. C. 2020; 9 (12): 3388-3399

  Abstract

  Structural proteins such as "suckerins" present promising avenues for fabricating functional materials. Suckerins are a family of naturally occurring block copolymer-type proteins that comprise the sucker ring teeth of cephalopods and are known to self-assemble into supramolecular networks of nanoconfined β-sheets. Here, we report the characterization and controllable, nanoscale self-assembly of suckerin-12 (S12). We characterize the impacts of salt, pH, and protein concentration on S12 solubility, secondary structure, and self-assembly. In doing so, we identify conditions for fabricating ∼100 nm nanoassemblies (NAs) with narrow size distributions. Finally, by installing a noncanonical amino acid (ncAA) into S12, we demonstrate the assembly of NAs that are covalently conjugated with a hydrophobic fluorophore and the ability to change self-assembly and β-sheet content by PEGylation. This work presents new insights into the biochemistry of suckerin-12 and demonstrates how ncAAs can be used to expedite and fine-tune the design of protein materials.

  View details for DOI 10.1021/acssynbio.0c00442

  View details for Web of Science ID 000607474600020

  View details for PubMedID 33201684

• Ribosome-mediated polymerization of long chain carbon and cyclic amino acids into peptides in vitro NATURE COMMUNICATIONS Lee, J., Schwarz, K. J., Kim, D., Moore, J. S., Jewett, M. C. 2020; 11 (1): 4304

  Abstract

  Ribosome-mediated polymerization of backbone-extended monomers into polypeptides is challenging due to their poor compatibility with the translation apparatus, which evolved to use α-L-amino acids. Moreover, mechanisms to acylate (or charge) these monomers to transfer RNAs (tRNAs) to make aminoacyl-tRNA substrates is a bottleneck. Here, we rationally design non-canonical amino acid analogs with extended carbon chains (γ-, δ-, ε-, and ζ-) or cyclic structures (cyclobutane, cyclopentane, and cyclohexane) to improve tRNA charging. We then demonstrate site-specific incorporation of these non-canonical, backbone-extended monomers at the N- and C- terminus of peptides using wild-type and engineered ribosomes. This work expands the scope of ribosome-mediated polymerization, setting the stage for new medicines and materials.

  View details for DOI 10.1038/s41467-020-18001-x

  View details for Web of Science ID 000567932900003

  View details for PubMedID 32855412

  View details for PubMedCentralID PMC7452890

• In vitro ribosome synthesis and evolution through ribosome display. Nature communications Hammerling, M. J., Fritz, B. R., Yoesep, D. J., Kim, D. S., Carlson, E. D., Jewett, M. C. 2020; 11 (1): 1108

  Abstract

  Directed evolution of the ribosome for expanded substrate incorporation and novel functions is challenging because the requirement of cell viability limits the mutations that can be made. Here we address this challenge by combining cell-free synthesis and assembly of translationally competent ribosomes with ribosome display to develop a fully in vitro methodology for ribosome synthesis and evolution (called RISE). We validate the RISE method by selecting active genotypes from a ~1.7 × 107 member library of ribosomal RNA (rRNA) variants, as well as identifying mutant ribosomes resistant to the antibiotic clindamycin from a library of ~4 × 103 rRNA variants. We further demonstrate the prevalence of positive epistasis in resistant genotypes, highlighting the importance of such interactions in selecting for new function. We anticipate that RISE will facilitate understanding of molecular translation and enable selection of ribosomes with altered properties.

  View details for DOI 10.1038/s41467-020-14705-2

  View details for PubMedID 32111839

• Expanding the limits of the second genetic code with ribozymes. Nature communications Lee, J., Schwieter, K. E., Watkins, A. M., Kim, D. S., Yu, H., Schwarz, K. J., Lim, J., Coronado, J., Byrom, M., Anslyn, E. V., Ellington, A. D., Moore, J. S., Jewett, M. C. 2019; 10 (1): 5097

  Abstract

  The site-specific incorporation of noncanonical monomers into polypeptides through genetic code reprogramming permits synthesis of bio-based products that extend beyond natural limits. To better enable such efforts, flexizymes (transfer RNA (tRNA) synthetase-like ribozymes that recognize synthetic leaving groups) have been used to expand the scope of chemical substrates for ribosome-directed polymerization. The development of design rules for flexizyme-catalyzed acylation should allow scalable and rational expansion of genetic code reprogramming. Here we report thesystematic synthesis of 37 substrates based on 4 chemically diverse scaffolds (phenylalanine, benzoic acid, heteroaromatic, and aliphatic monomers) with different electronic and steric factors. Of these substrates, 32 were acylated onto tRNA and incorporated into peptides by in vitro translation. Based on the design rules derived from this expanded alphabet, we successfully predicted the acylation of 6 additional monomers that could uniquely be incorporated into peptides and direct N-terminal incorporation of an aldehyde group for orthogonal bioconjugation reactions.

  View details for DOI 10.1038/s41467-019-12916-w

  View details for PubMedID 31704912

• Engineered ribosomes with tethered subunits for expanding biological function. Nature communications Carlson, E. D., d'Aquino, A. E., Kim, D. S., Fulk, E. M., Hoang, K., Szal, T., Mankin, A. S., Jewett, M. C. 2019; 10 (1): 3920

  Abstract

  Ribo-T is a ribosome with covalently tethered subunits where core 16S and 23S ribosomal RNAs form a single chimeric molecule. Ribo-T makes possible a functionally orthogonal ribosome-mRNA system in cells. Unfortunately, use of Ribo-T has been limited because of low activity of its original version. Here, to overcome this limitation, we use an evolutionary approach to select new tether designs that are capable of supporting faster cell growth and increased protein expression. Further, we evolve new orthogonal Ribo-T/mRNA pairs that function in parallel with, but independent of, natural ribosomes and mRNAs, increasing the efficiency of orthogonal protein expression. The Ribo-T with optimized designs is able to synthesize a diverse set of proteins, and can also incorporate multiple non-canonical amino acids into synthesized polypeptides. The enhanced Ribo-T designs should be useful for exploring poorly understood functions of the ribosome and engineering ribosomes with altered catalytic properties.

  View details for DOI 10.1038/s41467-019-11427-y

  View details for PubMedID 31477696

• Computational design of three-dimensional RNA structure and function. Nature nanotechnology Yesselman, J. D., Eiler, D. n., Carlson, E. D., Gotrik, M. R., d'Aquino, A. E., Ooms, A. N., Kladwang, W. n., Carlson, P. D., Shi, X. n., Costantino, D. A., Herschlag, D. n., Lucks, J. B., Jewett, M. C., Kieft, J. S., Das, R. n. 2019

  Abstract

  RNA nanotechnology seeks to create nanoscale machines by repurposing natural RNA modules. The field is slowed by the current need for human intuition during three-dimensional structural design. Here, we demonstrate that three distinct problems in RNA nanotechnology can be reduced to a pathfinding problem and automatically solved through an algorithm called RNAMake. First, RNAMake discovers highly stable single-chain solutions to the classic problem of aligning a tetraloop and its sequence-distal receptor, with experimental validation from chemical mapping, gel electrophoresis, solution X-ray scattering and crystallography with 2.55 Å resolution. Second, RNAMake automatically generates structured tethers that integrate 16S and 23S ribosomal RNAs into single-chain ribosomal RNAs that remain uncleaved by ribonucleases and assemble onto messenger RNA. Third, RNAMake enables the automated stabilization of small-molecule binding RNAs, with designed tertiary contacts that improve the binding affinity of the ATP aptamer and improve the fluorescence and stability of the Spinach RNA in cell extracts and in living Escherichia coli cells.

  View details for DOI 10.1038/s41565-019-0517-8

  View details for PubMedID 31427748

• Cell-free biomanufacturing CURRENT OPINION IN CHEMICAL ENGINEERING Bundy, B. C., Hunt, J., Jewett, M. C., Swartz, J. R., Wood, D. W., Frey, D. D., Rao, G. 2018; 22: 177–83

  View details for DOI 10.1016/j.coche.2018.10.003

  View details for Web of Science ID 000454025900023

• <i>Neisseria gonorrhoeae</i> Exposed to Sublethal Levels of Hydrogen Peroxide Mounts a Complex Transcriptional Response MSYSTEMS Quillin, S. J., Hockenberry, A. J., Jewett, M. C., Seifert, H. 2018; 3 (5)

  Abstract

  Neisseria gonorrhoeae mounts a substantial transcriptional program in response to hydrogen peroxide (HP), a prominent reactive oxygen species (ROS) encountered during infection. We tested which strain FA1090 genes show differential transcript abundance in response to sublethal amounts of HP to differentiate HP-responsive signaling from widespread cellular death and dysregulation. RNA sequencing (RNA-Seq) revealed that 150 genes were significantly upregulated and 143 genes downregulated following HP exposure. We annotated HP-responsive operons and all transcriptional start sites (TSSs) and identified which TSSs responded to HP treatment. We compared the HP responses and other previously reported genes and found only partial overlapping of other regulatory networks, indicating that the response to HP involves multiple biological functions. Using a representative subset of responsive genes, we validated the RNA-Seq results and found that the HP transcriptome was similar to that of sublethal organic peroxide. None of the genes in the representative subset, however, responded to sublethal levels of HOCl or O2 -. These results support the idea that N. gonorrhoeae may use variations in HP levels as a signal for different stages of infection. IMPORTANCE The strict human pathogen Neisseria gonorrhoeae is the only causative agent of the sexually transmitted disease gonorrhea. This bacterium encounters hydrogen peroxide produced from host cells during infection, but the organism survives in the presence of this antimicrobial agent. This work shows that the bacterium responds to hydrogen peroxide by regulating the expression of many genes involved in multiple processes.

  View details for DOI 10.1128/mSystems.00156-18

  View details for Web of Science ID 000449523700018

  View details for PubMedID 30320218

  View details for PubMedCentralID PMC6172773

• How many human proteoforms are there? Nature chemical biology Aebersold, R. n., Agar, J. N., Amster, I. J., Baker, M. S., Bertozzi, C. R., Boja, E. S., Costello, C. E., Cravatt, B. F., Fenselau, C. n., Garcia, B. A., Ge, Y. n., Gunawardena, J. n., Hendrickson, R. C., Hergenrother, P. J., Huber, C. G., Ivanov, A. R., Jensen, O. N., Jewett, M. C., Kelleher, N. L., Kiessling, L. L., Krogan, N. J., Larsen, M. R., Loo, J. A., Ogorzalek Loo, R. R., Lundberg, E. n., MacCoss, M. J., Mallick, P. n., Mootha, V. K., Mrksich, M. n., Muir, T. W., Patrie, S. M., Pesavento, J. J., Pitteri, S. J., Rodriguez, H. n., Saghatelian, A. n., Sandoval, W. n., Schlüter, H. n., Sechi, S. n., Slavoff, S. A., Smith, L. M., Snyder, M. P., Thomas, P. M., Uhlén, M. n., Van Eyk, J. E., Vidal, M. n., Walt, D. R., White, F. M., Williams, E. R., Wohlschlager, T. n., Wysocki, V. H., Yates, N. A., Young, N. L., Zhang, B. n. 2018; 14 (3): 206–14

  Abstract

  Despite decades of accumulated knowledge about proteins and their post-translational modifications (PTMs), numerous questions remain regarding their molecular composition and biological function. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Here, we outline what we know from current databases and measurement strategies including mass spectrometry-based proteomics. In doing so, we examine prevailing notions about the number of modifications displayed on human proteins and how they combine to generate the protein diversity underlying health and disease. We frame central issues regarding determination of protein-level variation and PTMs, including some paradoxes present in the field today. We use this framework to assess existing data and to ask the question, "How many distinct primary structures of proteins (proteoforms) are created from the 20,300 human genes?" We also explore prospects for improving measurements to better regularize protein-level biology and efficiently associate PTMs to function and phenotype.

  View details for PubMedID 29443976

• A Pressure Test to Make 10 Molecules in 90 Days: External Evaluation of Methods to Engineer Biology. Journal of the American Chemical Society Casini, A. n., Chang, F. Y., Eluere, R. n., King, A. M., Young, E. M., Dudley, Q. M., Karim, A. n., Pratt, K. n., Bristol, C. n., Forget, A. n., Ghodasara, A. n., Warden-Rothman, R. n., Gan, R. n., Cristofaro, A. n., Borujeni, A. E., Ryu, M. H., Li, J. n., Kwon, Y. C., Wang, H. n., Tatsis, E. n., Rodriguez-Lopez, C. n., O'Connor, S. n., Mdema, M. H., Fischbach, M. A., Jewett, M. C., Voigt, C. n., Gordon, D. B. 2018

  Abstract

  Centralized facilities for genetic engineering, or "biofoundries", offer the potential to design organisms to address emerging needs in medicine, agriculture, industry, and defense. The field has seen rapid advances in technology, but it is difficult to gauge current capabilities or identify gaps across projects. To this end, our foundry was assessed via a timed "pressure test", in which 3 months were given to build organisms to produce 10 molecules unknown to us in advance. By applying a diversity of new approaches, we produced the desired molecule or a closely related one for six out of 10 targets during the performance period and made advances toward production of the others as well. Specifically, we increased the titers of 1-hexadecanol, pyrrolnitrin, and pacidamycin D, found novel routes to the enediyne warhead underlying powerful antimicrobials, established a cell-free system for monoterpene production, produced an intermediate toward vincristine biosynthesis, and encoded 7802 individually retrievable pathways to 540 bisindoles in a DNA pool. Pathways to tetrahydrofuran and barbamide were designed and constructed, but toxicity or analytical tools inhibited further progress. In sum, we constructed 1.2 Mb DNA, built 215 strains spanning five species ( Saccharomyces cerevisiae, Escherichia coli, Streptomyces albidoflavus, Streptomyces coelicolor, and Streptomyces albovinaceus), established two cell-free systems, and performed 690 assays developed in-house for the molecules.

  View details for PubMedID 29480720

• Mapping Condition-Dependent Regulation of Lipid Metabolism in <i>Saccharomyces cerevisiae</i> G3-GENES GENOMES GENETICS Jewett, M. C., Workman, C. T., Nookaew, I., Pizarro, F. A., Agosin, E., Hellgren, L. I., Nielsen, J. 2013; 3 (11): 1979-1995

  Abstract

  Lipids play a central role in cellular function as constituents of membranes, as signaling molecules, and as storage materials. Although much is known about the role of lipids in regulating specific steps of metabolism, comprehensive studies integrating genome-wide expression data, metabolite levels, and lipid levels are currently lacking. Here, we map condition-dependent regulation controlling lipid metabolism in Saccharomyces cerevisiae by measuring 5636 mRNAs, 50 metabolites, 97 lipids, and 57 (13)C-reaction fluxes in yeast using a three-factor full-factorial design. Correlation analysis across eight environmental conditions revealed 2279 gene expression level-metabolite/lipid relationships that characterize the extent of transcriptional regulation in lipid metabolism relative to major metabolic hubs within the cell. To query this network, we developed integrative methods for correlation of multi-omics datasets that elucidate global regulatory signatures. Our data highlight many characterized regulators of lipid metabolism and reveal that sterols are regulated more at the transcriptional level than are amino acids. Beyond providing insights into the systems-level organization of lipid metabolism, we anticipate that our dataset and approach can join an emerging number of studies to be widely used for interrogating cellular systems through the combination of mathematical modeling and experimental biology.

  View details for DOI 10.1534/g3.113.006601

  View details for Web of Science ID 000327241900008

  View details for PubMedID 24062529

  View details for PubMedCentralID PMC3815060

• Precise Manipulation of Chromosomes in Vivo Enables Genome-Wide Codon Replacement SCIENCE Isaacs, F. J., Carr, P. A., Wang, H. H., Lajoie, M. J., Sterling, B., Kraal, L., Tolonen, A. C., Gianoulis, T. A., Goodman, D. B., Reppas, N. B., Emig, C. J., Bang, D., Hwang, S. J., Jewett, M. C., Jacobson, J. M., Church, G. M. 2011; 333 (6040): 348-353

  Abstract

  We present genome engineering technologies that are capable of fundamentally reengineering genomes from the nucleotide to the megabase scale. We used multiplex automated genome engineering (MAGE) to site-specifically replace all 314 TAG stop codons with synonymous TAA codons in parallel across 32 Escherichia coli strains. This approach allowed us to measure individual recombination frequencies, confirm viability for each modification, and identify associated phenotypes. We developed hierarchical conjugative assembly genome engineering (CAGE) to merge these sets of codon modifications into genomes with 80 precise changes, which demonstrate that these synonymous codon substitutions can be combined into higher-order strains without synthetic lethal effects. Our methods treat the chromosome as both an editable and an evolvable template, permitting the exploration of vast genetic landscapes.

  View details for DOI 10.1126/science.1205822

  View details for Web of Science ID 000292732000045

  View details for PubMedID 21764749

• Reconstruction of the yeast Snf1 kinase regulatory network reveals its role as a global energy regulator MOLECULAR SYSTEMS BIOLOGY Usaite, R., Jewett, M. C., Oliveira, A., Yates, J. R., Olsson, L., Nielsen, J. 2009; 5: 319

  Abstract

  Highly conserved among eukaryotic cells, the AMP-activated kinase (AMPK) is a central regulator of carbon metabolism. To map the complete network of interactions around AMPK in yeast (Snf1) and to evaluate the role of its regulatory subunit Snf4, we measured global mRNA, protein and metabolite levels in wild type, Deltasnf1, Deltasnf4, and Deltasnf1Deltasnf4 knockout strains. Using four newly developed computational tools, including novel DOGMA sub-network analysis, we showed the benefits of three-level ome-data integration to uncover the global Snf1 kinase role in yeast. We for the first time identified Snf1's global regulation on gene and protein expression levels, and showed that yeast Snf1 has a far more extensive function in controlling energy metabolism than reported earlier. Additionally, we identified complementary roles of Snf1 and Snf4. Similar to the function of AMPK in humans, our findings showed that Snf1 is a low-energy checkpoint and that yeast can be used more extensively as a model system for studying the molecular mechanisms underlying the global regulation of AMPK in mammals, failure of which leads to metabolic diseases.

  View details for DOI 10.1038/msb.2009.67

  View details for Web of Science ID 000272308900001

  View details for PubMedID 19888214

  View details for PubMedCentralID PMC2795470

• Linking high-resolution metabolic flux phenotypes and transcriptional regulation in yeast modulated by the global regulator Gcn4p PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Moxley, J. F., Jewett, M. C., Antoniewicz, M. R., Villas-Boas, S. G., Alper, H., Wheeler, R. T., Tong, L., Hinnebusch, A. G., Ideker, T., Nielsen, J., Stephanopoulos, G. 2009; 106 (16): 6477-6482

  Abstract

  Genome sequencing dramatically increased our ability to understand cellular response to perturbation. Integrating system-wide measurements such as gene expression with networks of protein-protein interactions and transcription factor binding revealed critical insights into cellular behavior. However, the potential of systems biology approaches is limited by difficulties in integrating metabolic measurements across the functional levels of the cell despite their being most closely linked to cellular phenotype. To address this limitation, we developed a model-based approach to correlate mRNA and metabolic flux data that combines information from both interaction network models and flux determination models. We started by quantifying 5,764 mRNAs, 54 metabolites, and 83 experimental (13)C-based reaction fluxes in continuous cultures of yeast under stress in the absence or presence of global regulator Gcn4p. Although mRNA expression alone did not directly predict metabolic response, this correlation improved through incorporating a network-based model of amino acid biosynthesis (from r = 0.07 to 0.80 for mRNA-flux agreement). The model provides evidence of general biological principles: rewiring of metabolic flux (i.e., use of different reaction pathways) by transcriptional regulation and metabolite interaction density (i.e., level of pairwise metabolite-protein interactions) as a key biosynthetic control determinant. Furthermore, this model predicted flux rewiring in studies of follow-on transcriptional regulators that were experimentally validated with additional (13)C-based flux measurements. As a first step in linking metabolic control and genetic regulatory networks, this model underscores the importance of integrating diverse data types in large-scale cellular models. We anticipate that an integrated approach focusing on metabolic measurements will facilitate construction of more realistic models of cellular regulation for understanding diseases and constructing strains for industrial applications.

  View details for DOI 10.1073/pnas.0811091106

  View details for Web of Science ID 000265506800012

  View details for PubMedID 19346491

  View details for PubMedCentralID PMC2672541

• Continued Protein Synthesis at Low [ATP] and [GTP] Enables Cell Adaptation during Energy Limitation JOURNAL OF BACTERIOLOGY Jewett, M. C., Miller, M. L., Chen, Y., Swartz, J. R. 2009; 191 (3): 1083-1091

  Abstract

  One of biology's critical ironies is the need to adapt to periods of energy limitation by using the energy-intensive process of protein synthesis. Although previous work has identified the individual energy-requiring steps in protein synthesis, we still lack an understanding of the dependence of protein biosynthesis rates on [ATP] and [GTP]. Here, we used an integrated Escherichia coli cell-free platform that mimics the intracellular, energy-limited environment to show that protein synthesis rates are governed by simple Michaelis-Menten dependence on [ATP] and [GTP] (K(m)(ATP), 27 +/- 4 microM; K(m)(GTP), 14 +/- 2 microM). Although the system-level GTP affinity agrees well with the individual affinities of the GTP-dependent translation factors, the system-level K(m)(ATP) is unexpectedly low. Especially under starvation conditions, when energy sources are limited, cells need to replace catalysts that become inactive and to produce new catalysts in order to effectively adapt. Our results show how this crucial survival priority for synthesizing new proteins can be enforced after rapidly growing cells encounter energy limitation. A diminished energy supply can be rationed based on the relative ATP and GTP affinities, and, since these affinities for protein synthesis are high, the cells can adapt with substantial changes in protein composition. Furthermore, our work suggests that characterization of individual enzymes may not always predict the performance of multicomponent systems with complex interdependencies. We anticipate that cell-free studies in which complex metabolic systems are activated will be valuable tools for elucidating the behavior of such systems.

  View details for DOI 10.1128/JB.00852-08

  View details for Web of Science ID 000262609200045

  View details for PubMedID 19028899

  View details for PubMedCentralID PMC2632092

• An integrated cell-free metabolic platform for protein production and synthetic biology MOLECULAR SYSTEMS BIOLOGY Jewett, M. C., Calhoun, K. A., Voloshin, A., Wuu, J. J., Swartz, J. R. 2008; 4

  Abstract

  Cell-free systems offer a unique platform for expanding the capabilities of natural biological systems for useful purposes, i.e. synthetic biology. They reduce complexity, remove structural barriers, and do not require the maintenance of cell viability. Cell-free systems, however, have been limited by their inability to co-activate multiple biochemical networks in a single integrated platform. Here, we report the assessment of biochemical reactions in an Escherichia coli cell-free platform designed to activate natural metabolism, the Cytomim system. We reveal that central catabolism, oxidative phosphorylation, and protein synthesis can be co-activated in a single reaction system. Never before have these complex systems been shown to be simultaneously activated without living cells. The Cytomim system therefore promises to provide the metabolic foundation for diverse ab initio cell-free synthetic biology projects. In addition, we describe an improved Cytomim system with enhanced protein synthesis yields (up to 1200 mg/l in 2 h) and lower costs to facilitate production of protein therapeutics and biochemicals that are difficult to make in vivo because of their toxicity, complexity, or unusual cofactor requirements.

  View details for DOI 10.1038/msb.2008.57

  View details for Web of Science ID 000260722900002

  View details for PubMedID 18854819

  View details for PubMedCentralID PMC2583083

• Growth temperature exerts differential physiological and transcriptional responses in laboratory and wine strains of <i>Saccharomyces cerevisiae</i> APPLIED AND ENVIRONMENTAL MICROBIOLOGY Pizarro, F. J., Jewett, M. C., Nielsen, J., Agosin, E. 2008; 74 (20): 6358-6368

  Abstract

  Laboratory strains of Saccharomyces cerevisiae have been widely used as a model for studying eukaryotic cells and mapping the molecular mechanisms of many different human diseases. Industrial wine yeasts, on the other hand, have been selected on the basis of their adaptation to stringent environmental conditions and the organoleptic properties that they confer to wine. Here, we used a two-factor design to study the responses of a standard laboratory strain, CEN.PK113-7D, and an industrial wine yeast strain, EC1118, to growth temperatures of 15 degrees C and 30 degrees C in nitrogen-limited, anaerobic, steady-state chemostat cultures. Physiological characterization revealed that the growth temperature strongly impacted the biomass yield of both strains. Moreover, we found that the wine yeast was better adapted to mobilizing resources for biomass production and that the laboratory yeast exhibited higher fermentation rates. To elucidate mechanistic differences controlling the growth temperature response and underlying adaptive mechanisms between the strains, DNA microarrays and targeted metabolome analysis were used. We identified 1,007 temperature-dependent genes and 473 strain-dependent genes. The transcriptional response was used to identify highly correlated gene expression subnetworks within yeast metabolism. We showed that temperature differences most strongly affect nitrogen metabolism and the heat shock response. A lack of stress response element-mediated gene induction, coupled with reduced trehalose levels, indicated that there was a decreased general stress response at 15 degrees C compared to that at 30 degrees C. Differential responses among strains were centered on sugar uptake, nitrogen metabolism, and expression of genes related to organoleptic properties. Our study provides global insight into how growth temperature affects differential physiological and transcriptional responses in laboratory and wine strains of S. cerevisiae.

  View details for DOI 10.1128/AEM.00602-08

  View details for Web of Science ID 000259985300024

  View details for PubMedID 18723660

  View details for PubMedCentralID PMC2570279

• Transcription factor control of growth rate dependent genes in <i>Saccharomyces cerevisiae</i>:: A three factor design BMC GENOMICS Fazio, A., Jewett, M. C., Daran-Lapujade, P., Mustacchi, R., Usaite, R., Pronk, J. T., Workman, C. T., Nielsen, J. 2008; 9: 341

  Abstract

  Characterization of cellular growth is central to understanding living systems. Here, we applied a three-factor design to study the relationship between specific growth rate and genome-wide gene expression in 36 steady-state chemostat cultures of Saccharomyces cerevisiae. The three factors we considered were specific growth rate, nutrient limitation, and oxygen availability.We identified 268 growth rate dependent genes, independent of nutrient limitation and oxygen availability. The transcriptional response was used to identify key areas in metabolism around which mRNA expression changes are significantly associated. Among key metabolic pathways, this analysis revealed de novo synthesis of pyrimidine ribonucleotides and ATP producing and consuming reactions at fast cellular growth. By scoring the significance of overlap between growth rate dependent genes and known transcription factor target sets, transcription factors that coordinate balanced growth were also identified. Our analysis shows that Fhl1, Rap1, and Sfp1, regulating protein biosynthesis, have significantly enriched target sets for genes up-regulated with increasing growth rate. Cell cycle regulators, such as Ace2 and Swi6, and stress response regulators, such as Yap1, were also shown to have significantly enriched target sets.Our work, which is the first genome-wide gene expression study to investigate specific growth rate and consider the impact of oxygen availability, provides a more conservative estimate of growth rate dependent genes than previously reported. We also provide a global view of how a small set of transcription factors, 13 in total, contribute to control of cellular growth rate. We anticipate that multi-factorial designs will play an increasing role in elucidating cellular regulation.

  View details for DOI 10.1186/1471-2164-9-341

  View details for Web of Science ID 000258235300001

  View details for PubMedID 18638364

  View details for PubMedCentralID PMC2500033

• Metabolic modeling of cell-free protein synthesis reactions. 229th National Meeting of the American-Chemical-Society (ACS) Calhoun, K. A., Varner, J., Jewett, M. C., Swartz, J. R. AMER CHEMICAL SOC. 2005: U194–U194

  View details for Web of Science ID 000228177701095

• Substrate replenishment extends protein synthesis with an in vitro translation system designed to mimic the cytoplasm BIOTECHNOLOGY AND BIOENGINEERING Jewett, M. C., Swartz, J. R. 2004; 87 (4): 465-472

  Abstract

  Cytoplasmic mimicry has recently led to the development of a novel method for cell-free protein synthesis called the "Cytomim" system. In vitro translation with this new system produced more than a 5-fold yield increase of chloramphenicol acetyl transferase (CAT) relative to a conventional method using pyruvate as an energy substrate. Factors responsible for activating enhanced protein yields, and causes leading to protein synthesis termination have been assessed in this new system. Enhanced yields were caused by the combination of three changes: growing the extract source cells on 2x YTPG media versus 2x YT, replacing polyethylene glycol with spermidine and putrescine, and reducing the magnesium concentration from conventional levels. Cessation of protein synthesis was primarily caused by depletion of cysteine, serine, CTP, and UTP. Substrate replenishment of consumed amino acids, CTP, and UTP extended the duration of protein synthesis to 24 h in fed-batch operation and produced 1.2 mg/mL of CAT. By also adding more T7 RNA polymerase and plasmid DNA, yields were further improved to 1.4 mg/mL of CAT. These results underscore the critical role that nucleotides play in the combined transcription-translation reaction and highlight the importance of understanding metabolic processes influencing substrate depletion.

  View details for DOI 10.1002/bit.20139

  View details for Web of Science ID 000223072500004

  View details for PubMedID 15286983

• Mimicking the Escherichia coli cytoplasmic environment activates long-lived and efficient cell-free protein synthesis BIOTECHNOLOGY AND BIOENGINEERING Jewett, M. C., Swartz, J. R. 2004; 86 (1): 19-26

  Abstract

  Cell-free translation systems generally utilize high-energy phosphate compounds to regenerate the adenosine triphosphate (ATP) necessary to drive protein synthesis. This hampers the widespread use and practical implementation of this technology in a batch format due to expensive reagent costs; the accumulation of inhibitory byproducts, such as phosphate; and pH change. To address these problems, a cell-free protein synthesis system has been engineered that is capable of using pyruvate as an energy source to produce high yields of protein. The "Cytomim" system, synthesizes chloramphenicol acetyltransferase (CAT) for up to 6 h in a batch reaction to yield 700 microg/mL of protein. By more closely replicating the physiological conditions of the cytoplasm of Escherichia coli, the Cytomim system provides a stable energy supply for protein expression without phosphate accumulation, pH change, exogenous enzyme addition, or the need for expensive high-energy phosphate compounds.

  View details for DOI 10.1002/bit.20026

  View details for Web of Science ID 000220196000003

  View details for PubMedID 15007837

• Using cell-free biology to study systems biology. 227th National Meeting of the American-Chemical Society Swartz, J. R., Calhoun, K. A., Jewett, M. C. AMER CHEMICAL SOC. 2004: U255–U255

  View details for Web of Science ID 000223655600964

• Systems approach to translation: Defining the protein production rate dependence on cell extract concentration. Jewett, M. C., Underwood, K. A., Swartz AMER CHEMICAL SOC. 2004: U131

  View details for Web of Science ID 000223655600603

• Rapid expression and purification of 100 nmol quantities of active protein using cell-free protein synthesis BIOTECHNOLOGY PROGRESS Jewett, M. C., Swartz, J. R. 2004; 20 (1): 102-109

  Abstract

  Two strategies for ATP regeneration during cell-free protein synthesis were applied to the large-scale production and single-column purification of active chloramphenicol acetyl transferase (CAT). Fed-batch reactions were performed on a 5-10 mL scale, approximately 2 orders of magnitude greater than the typical reaction volume. The pyruvate oxidase system produced 104 nmol of active CAT in a 5 mL reaction over the course of 5 h. The PANOx system produced 261 +/- 42 nmol, about 7 mg, of active CAT in a 10 mL reaction over the course of 4 h. The reaction product was purified to apparent homogeneity with approximately 70% yield by a simple affinity chromatography adsorption and elution. To our knowledge, this is the largest amount of actively expressed protein to be reported in a simple, fed-batch cell-free protein synthesis reaction.

  View details for DOI 10.1021/bp0341693

  View details for Web of Science ID 000188861300014

  View details for PubMedID 14763830

• Cell-free protein synthesis with prokaryotic combined transcription-translation. Methods in molecular biology (Clifton, N.J.) Swartz, J. R., Jewett, M. C., Woodrow, K. A. 2004; 267: 169-182

  Abstract

  Cell-free biology exploits and studies complex biological processes in a controlled environment without intact cells. One model system is prokaryotic cell-free protein synthesis. This technology offers an attractive and convenient approach to produce properly folded recombinant DNA (rDNA) proteins on a laboratory scale, screen PCR fragment libraries in a high-throughput format, express pharmaceutical proteins, incorporate labeled or unnatural amino acids into proteins, and activate microbial physiology to allow for investigation of biological systems. We describe the preparation of materials necessary for the expression, quantification, and purification of rDNA proteins from active Escherichia coli extracts.

  View details for PubMedID 15269424