Michael Christopher Jewett

All Publications

• 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

• 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

• 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

• 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

• 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

• 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

• 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

• 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

• 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

• 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