Biology subjects

Jewett, M. C.

Publications and source records attributed to Jewett, M. C..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Fluorescent minihelix assay for translation quantification in high-throughput cell-free systems

We report the use of an optimized tetra-cysteine minihelix both as a fusion protein and as a standalone reporter in vitro with the Flash dye to study cell-free protein expression dynamics and engineered ribosome activity. The fluorescent product can be detected and quantified via its characteristic emission spectrum in a standard 96/384-well plate reader, RT-qPCR system, or gel electrophoresis. The fluorescent reporter helix is short enough to be encoded on a primer pair and can tag any protein of interest via PCR. Both tagged protein or standalone reporter can be detected in real time during and in terminal cell-free expression reactions, or in gel, without the need for staining. The fluorescent signal is stable and linearly correlates with protein concentration, thus is suitable for product quantification. Finally, we demonstrate how this reporter can be used for future efforts in engineering in vitro translation systems. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/562088v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@4eb383org.highwire.dtl.DTLVardef@eb8391org.highwire.dtl.DTLVardef@ae08forg.highwire.dtl.DTLVardef@1cd58a_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

Improving cell-free expression of membrane proteins by tuning ribosome co-translational membrane association and nascent chain aggregation

Cell-free gene expression (CFE) systems are powerful tools for transcribing and translating genes outside of a living cell. Given their diverse roles in nature, synthesis of membrane proteins is of particular interest, but their yield in CFE is substantially lower than for soluble protein. In this paper, we study factors that affect the cell-free synthesis of membrane proteins and develop a quantitative kinetic model of their production. We identify that stalling of membrane protein translation on the ribosome is a strong predictor of membrane protein synthesis and creates a negative feedback loop in which stalled peptide sequences quench ribosome activity through aggregation between the ribosome nascent chains. Synthesis can be improved by the addition of lipid membranes which incorporate protein nascent chains and, therefore, kinetically competes with aggregation. Using both quantitative modeling and experiment, we show that the balance between peptide-membrane association and peptide aggregation rates determines the total yield of synthesized membrane protein. We then demonstrate that this balance can be shifted by altering membrane composition or the protein N-terminal domain sequence. Based on these findings, we define a membrane protein expression score that can be used to rationalize the engineering of N-terminal domain sequences both of a native and computationally designed membrane proteins produced through CFE.

synthetic biology↗

bioRxiv2023

At-home, cell-free synthetic biology education modules for transcriptional regulation and environmental water quality monitoring

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.

synthetic biology↗

bioRxiv2022

Point-of-care peptide hormone production enabled by cell-free protein synthesis

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. Towards 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(R) affinity purification and on-bead SUMO protease cleavage yields 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 this platform could be used to manufacture valuable peptide hormone drugs at the point-of-care in resource-limited settings.

synthetic biology↗

bioRxiv2022

A low-cost recombinant glycoconjugate vaccine confers immunogenicity and protection against enterotoxigenic Escherichia coli infections in mice

Enterotoxigenic Escherichia coli (ETEC) is the primary etiologic agent of travelers 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 significantly reduces mortality and morbidity associated with moderate to severe diarrhea among children under the age of 5 years remains an unmet aspirational goal. Here, we describe robust, cost-effective biosynthetic routes that leverage glycoengineered strains of non-pathogenic Escherichia 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 confers protection against ETEC infection in mice. 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.

synthetic biology↗

bioRxiv2022

Orthogonal tRNA Expression using Endogenous Machinery in Cell-Free Systems

A wide variety of non-canonical amino acids (ncAAs) can be incorporated into proteins through the coordinated action of a stop codon suppressing tRNA and aminoacyl-tRNA synthetase. However, methods to discover and characterize suppressor tRNAs are generally lacking. In this work, we show that cell-free systems can express functional suppressor tRNAs using endogenous machinery and characterize their activity. This method is compatible with widely used orthogonal tRNAs, such as the Methanocaldococus jannaschii tyrosyl tRNA, the Methanosarcina barkeri pyrrolysyl tRNA, the Methanomethylophilus Alvus pyrrolysyl tRNA, and an engineered Int pyrroysyl tRNA. Modifying the workflow to evaluate TAA suppression revealed that the M. jannaschii and M. alvus are highly functional TAA suppressors in cell-free systems. Finally, we show that we can express two distinct tRNAs simultaneously, enabling the incorporation of multiple, distinct ncAAs. In total, our work shows that cell-free systems are useful platforms to express and characterize tRNAs.

synthetic biology↗

bioRxiv2022

Dynamic Kinetic Models Capture Cell-Free Metabolism for Improved Butanol Production

Cell-free systems are useful tools for prototyping metabolic pathways and optimizing the production of various bioproducts. Mechanistically-based kinetic models are uniquely suited to analyze dynamic experimental data collected from cell-free systems and provide vital qualitative insight. However, to date, dynamic kinetic models have not been applied with rigorous biological constraints or trained on adequate experimental data to the degree that they would give high confidence in predictions and broadly demonstrate the potential for widespread use of such kinetic models. In this work, we construct a large-scale dynamic model of cell-free metabolism with the goal of understanding and optimizing butanol production in a cell-free system. Using a novel combination of parameterization methods, the resultant model captures experimental metabolite measurements across two experimental conditions for nine metabolites at timepoints between 0 and 24 hours. We present analysis of the model predictions, provide recommendations for butanol optimization, and identify the aldehyde/alcohol dehydrogenase as the primary bottleneck in butanol production. Sensitivity analysis further reveals the extent to which various parameters are constrained, and our approach for probing valid parameter ranges can be applied to other modeling efforts.

synthetic biology↗

bioRxiv2022

A low-cost, thermostable, cell-free protein synthesis platform for on demand production of conjugate vaccines

Cell-free protein synthesis systems that can be lyophilized for long-term, nonrefrigerated storage and transportation have the potential to enable decentralized biomanufacturing. However, increased thermostability and decreased reaction cost are necessary for further technology adoption. Here, we identify maltodextrin as an additive to cell-free reactions that can act as both a lyoprotectant to increase thermostability, as well as a lowcost energy substrate. As a model, we apply optimized formulations to produce conjugate vaccines for ~$0.50 per dose after storage at room temperature or 37 {degrees}C for up to 4 weeks and ~$1.00 per dose after storage at 50 {degrees}C for up to 4 weeks. We show that these conjugates generate bactericidal antibodies against enterotoxigenic E. coli (ETEC) O78 O-polysaccharide, a pathogen responsible for diarrheal disease, in immunized mice. We anticipate that our lowcost, thermostable cell-free glycoprotein synthesis system will enable new models of medicine biosynthesis and distribution that bypass cold-chain requirements.

synthetic biology↗

bioRxiv2022

Ribosome display of N-linked glycoproteins in cell-free extracts

Ribosome display is a powerful in vitro method for the selection and directed evolution of proteins expressed from combinatorial libraries. However, because ribosome display is typically performed with standard in vitro translation reagents, the ability to display proteins with complex post-translational modifications such as glycosylation is limited. To address this technological gap, here we developed a set of complementary methods for producing stalled ribosome complexes that displayed asparagine-linked (N-linked) glycoproteins in conformations amenable to downstream functional and glyco-structural interrogation. The ability to generate glycosylated ribosome-nascent chain (glycoRNC) complexes was enabled by integrating SecM-mediated translation arrest with methods for cell-free synthesis of N-glycoproteins. This integration yielded a novel capability for translating and displaying target proteins modified efficiently and site-specifically with different N-glycan structures. Moreover, the encoding mRNAs remained stably attached to stalled ribosomes both before and after biopanning, thereby providing the genotype- glycophenotype link between an arrested glycoprotein and its RNA message. We anticipate that our method will enable selection and evolution of N-linked glycoproteins with advantageous biological and biophysical properties.

synthetic biology↗

bioRxiv2022

Computationally-guided design and selection of ribosomal active site mutants with high activity

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, 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 may serve as a powerful tool for high-resolution rRNA design.

synthetic biology↗

bioRxiv2021

Improving genomically recoded Escherichia coli for the production of proteins containing non-canonical amino acids

A genomically recoded Escherichia coli strain that lacks all amber codons and release factor 1 (C321.{Delta}A) enables efficient genetic encoding of chemically diverse, non-canonical amino acids (ncAAs) into proteins. While C321.{Delta}A has opened new opportunities in chemical and synthetic biology, this strain has not been optimized for protein production, limiting its utility in widespread industrial and academic applications. To address this limitation, we describe the construction of a series of genomically recoded organisms that are optimized for cellular protein production. We demonstrate that the functional deactivation of nucleases (e.g., rne, endA) and proteases (e.g., lon) increases production of wild-type superfolder green fluorescent protein (sfGFP) and sfGFP containing two ncAAs up to [~]5-fold. Additionally, we introduce a genomic IPTG-inducible T7 RNA polymerase (T7RNAP) cassette into these strains. Using an optimized platform, we demonstrated the ability to introduce 2 identical N6-(propargyloxycarbonyl)-L-Lysine residues site specifically into sfGFP with a 17-fold improvement in production relative to the parent. We envision that our library of organisms will provide the community with multiple options for increased expression of proteins with new and diverse chemistries.

synthetic biology↗

bioRxiv2021

A high-throughput, automated, cell-free expression and screening platform for antibody discovery

Antibody discovery is bottlenecked by the individual expression and evaluation of antigen-specific hits. Here, we address this gap by developing an automated workflow combining cell-free DNA template generation, protein synthesis, and high-throughput binding measurements of antibody fragments in a process that takes hours rather than weeks. We apply this workflow to 119 published SARS-CoV-2 neutralizing antibodies and demonstrate rapid identification of the most potent antibody candidates.

synthetic biology↗

bioRxiv2021

Vertex protein PduN tunes encapsulated pathway performance by dictating bacterial metabolosome morphology

Engineering subcellular organization in microbes shows great promise in addressing bottlenecks in metabolic engineering efforts; however, rules guiding selection of an organization strategy or platform are lacking. Here, we study compartment morphology as a factor in mediating encapsulated pathway performance. Using the 1,2-propanediol utilization microcompartment (Pdu MCP) system from Salmonella enterica serovar Typhimurium LT2, we find that we can shift the morphology of this protein nanoreactor from polyhedral to tubular by removing vertex protein PduN. Analysis of the metabolic function between these Pdu microtubes (MTs) shows that they provide a diffusional barrier capable of shielding the cytosol from a toxic pathway intermediate, similar to native MCPs. However, kinetic modeling suggests that the different surface area to volume ratios of MCP and MT structures alters encapsulated pathway performance. Finally, we report a microscopy-based assay that permits rapid assessment of Pdu MT formation to enable future engineering efforts on these structures.

bioengineering↗

bioRxiv2021

Linking the Salmonella enterica 1,2-propanediol utilization bacterial microcompartment shell to the enzymatic core via the shell protein PduB

Bacterial microcompartments (MCPs) are protein-based organelles that house the enzymatic machinery for metabolism of niche carbon sources, allowing enteric pathogens to outcompete native microbiota during host colonization. While much progress has been made toward understanding MCP biogenesis, questions still remain regarding the mechanism by which core MCP enzymes are enveloped within the MCP protein shell. Here we explore the hypothesis that the shell protein PduB is responsible for linking the shell of the 1,2-propanediol utilization (Pdu) MCP from Salmonella enterica serovar Typhimurium LT2 to its enzymatic core. Using fluorescent reporters, we demonstrate that all members of the Pdu enzymatic core are encapsulated in Pdu MCPs. We also demonstrate that PduB is the sole protein responsible for linking the entire Pdu enzyme core to the MCP shell. Using MCP purifications, transmission electron microscopy, and fluorescence microscopy we find that shell assembly can be decoupled from the enzymatic core, as apparently empty MCPs are formed in Salmonella strains lacking PduB. Mutagenesis studies also reveal that PduB is incorporated into the Pdu MCP shell via a conserved, lysine-mediated hydrogen bonding mechanism. Finally, growth assays and systems-level pathway modeling reveal that unencapsulated pathway performance is strongly impacted by enzyme concentration, highlighting the importance of minimizing polar effects when conducting these functional assays. Together, these results provide insight into the mechanism of enzyme encapsulation within Pdu MCPs and demonstrate that the process of enzyme encapsulation and shell assembly are separate processes in this system, a finding that will aid future efforts to understand MCP biogenesis. ImportanceMCPs are unique, genetically encoded organelles used by many bacteria to survive in resource-limited environments. There is significant interest in understanding the biogenesis and function of these organelles, both as potential antibiotic targets in enteric pathogens and also as useful tools for overcoming metabolic engineering bottlenecks. However, the mechanism by which these organelles are formed natively is still not completely understood. Here we provide evidence of a potential mechanism inS. enterica by which a single protein, PduB, links the MCP shell and metabolic core. This finding is critical for those seeking to disrupt MCPs during pathogenic infections or for those seeking to harness MCPs as nanobioreactors in industrial settings.

synthetic biology↗

bioRxiv2021

Development of a Freeze-Dried CRISPR-Cas12 Sensor for Detecting Wolbachia in the Secondary Science Classroom

Training the future synthetic biology workforce requires opportunity and exposure to biotechnology concepts and activities in secondary education. Detecting Wolbachia bacteria in arthropods using PCR has become a common way for secondary students to investigate and apply DNA technology in the science classroom. Despite this framework, cutting-edge biotechnologies like CRISPR-based diagnostics have yet to be widely implemented in the classroom. To address this gap, we present a freeze-dried CRISPR-Cas12 sensing reaction to complement traditional DNA technology education and teach synthetic biology concepts. The reactions accurately detect Wolbachia from arthropod-derived PCR samples in under 2 hours and can be stored at room temperature for over a month without appreciable degradation. The reactions are easy-to-use and cost less than $40 to implement for a classroom of 22 students including the cost of reusable equipment. We see this technology as an accessible way to incorporate synthetic biology education into existing biology curriculum, which will expand biology educational opportunities in science, technology, engineering, and mathematics (STEM) education.

synthetic biology↗

bioRxiv2021

Community science designed ribosomes with beneficial phenotypes

Functional design of ribosomes with mutant ribosomal RNA (rRNA) could expand opportunities for understanding molecular translation, building cells from the bottom-up, and engineering ribosomes with altered capabilities. However, such efforts have been 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 developed 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 applied 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 new insights into ribosome rRNA sequence-function relationships, with implications for synthetic biology.

synthetic biology↗

bioRxiv2021

Spacer2PAM: A computational framework for identification of functional PAM sequences for endogenous CRISPR systems

RNA-guided nucleases from clustered regularly interspaced short palindromic repeats (CRISPR) systems expand opportunities for precise, targeted genome modification. Endogenous CRISPR systems in many bacteria and archaea are particularly attractive to circumvent expression, functionality, and unintended activity hurdles posed by heterologous CRISPR effectors. However, each CRISPR system recognizes a unique set of PAM sequences, which requires extensive screening of randomized DNA libraries. This challenge makes it difficult to develop endogenous CRISPR systems, especially in organisms that are slow-growing or have transformation idiosyncrasies. To address this limitation, we present Spacer2PAM, an easy-to-use, easy-to-interpret R package built to identify potential PAM sequences for any CRISPR system given its corresponding CRISPR array as input. Spacer2PAM can be used in "Quick" mode to generate a single PAM prediction that is likely to be functional or in "Comprehensive" mode to inform targeted, unpooled PAM libraries small enough to screen in difficult to transform organisms. We demonstrate Spacer2PAM by predicting PAM sequences for industrially relevant organisms and experimentally identifying seven PAM sequences that mediate interference from the Spacer2PAM-predicted PAM library for the type I-B CRISPR system from Clostridium autoethanogenum. We anticipate that Spacer2PAM will facilitate the use of endogenous CRISPR systems for industrial biotechnology and synthetic biology.

synthetic biology↗

bioRxiv2020

Characterizing and controlling nanoscale self-assembly of suckerin-12

Structural proteins such as the "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 {beta}-sheets. Here, we report characterization and controllable, nanoscale self-assembly of suckerin-12 (S12). We characterize 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 non-canonical 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 {beta}-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.

bioengineering↗