Biology subjects

Jewett, M. C.

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

At least 55 records · Page 3Linked to original sources

bioRxiv2020

Increasing cell-free gene expression yields from linear templates in Escherichia coli and Vibrio natriegens extracts by using DNA-binding proteins

In crude extract-based cell-free protein synthesis (CFPS), DNA templates are transcribed and translated into functional proteins. Although linear expression templates (LETs) are less laborious and expensive to generate, plasmid templates are often desired over PCR-generated LETs due to increased stability and protection against exonucleases present in the extract of the reaction. Here we demonstrate that addition of a dsDNA-binding protein to the CFPS reaction, termed single-chain Cro protein (scCro), achieves terminal protection of LETs. This CroP-LET (scCro-based Protection of LET) method effectively increases sfGFP expression levels from LETs in Escherichia coli CFPS reactions by 6-fold. Our yields are comparable to other strategies that provide chemical and enzymatic DNA stabilization in E. coli CFPS. Notably, we also report that the CroP-LET method successfully enhanced yields in CFPS platforms derived from non-model organisms. Our results show that CroP-LET increased sfGFP yields by 18-fold in the Vibrio natriegens CFPS platform. With the fast-expanding applications of CFPS platforms, this method provides a practical and generalizable solution to protect linear expression DNA templates.

synthetic biology↗

bioRxiv2020

Modular cell-free expression plasmids to accelerate biological design in cells

Industrial biotechnology aims to produce high-value products from renewable resources. This can be challenging because model microorganisms--organisms that are easy to use like Escherichia coli--often lack the machinery required to utilize desired feedstocks like lignocellulosic biomass or syngas. Non-model organisms, such as Clostridium, are industrially proven and have the desired metabolic features but have several hurdles to mainstream use. Namely, these species grow more slowly than conventional laboratory microbes and genetic tools for engineering them are far less prevalent. To address these hurdles for accelerating cellular design, cell-free synthetic biology has emerged as an approach for characterizing non-model organisms and rapidly testing metabolic pathways in vitro. Unfortunately, cell-free systems can require specialized DNA architectures with minimal regulation that are not compatible with cellular expression. In this work, we develop a modular vector system that allows for T7 expression of desired enzymes for cell-free expression and direct Golden Gate assembly into Clostridium expression vectors. Utilizing the Joint Genome Institutes DNA Synthesis Community Science Program, we designed and synthesized these plasmids and genes required for our projects allowing us to shuttle DNA easily between our in vitro and in vivo experiments. We next validated that these vectors were sufficient for cell-free expression of enzymes, performing on par with the previous state-of-the-art. Lastly, we demonstrated automated six-part DNA assemblies for C. autoethanogenum expression with efficiencies ranging from 68-90%. We anticipate this system of plasmids will enable a framework for facile testing of biosynthetic pathways in vitro and in vivo by shortening development cycles.

synthetic biology↗

bioRxiv2020

Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles

Cell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for accelerating the design of cellular function, on-demand biomanufacturing, portable diagnostics, and educational kits. Many essential biological processes that could endow CFE systems with desired functions, such as protein glycosylation, rely on the activity of membrane-bound components. However, without the use of synthetic membrane mimics, activating membrane-dependent functionality in bacterial CFE systems remains largely unstudied. Here, we address this gap by characterizing native, cell-derived membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membranebound machinery. We first use nanocharacterization techniques to show that lipid vesicles in CFE extracts are tens to hundreds of nanometers across, and on the order of ~3x1012 particles/mL. We then determine how extract processing methods, such as post-lysis centrifugation, can be used to modulate concentrations of membrane vesicles in CFE systems. By tuning these methods, we show that increasing the number of vesicle particles to ~7x1012 particles/mL can be used to increase concentrations of heterologous membrane protein cargo expressed prior to lysis. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving N-linked and O-linked glycoprotein synthesis. We anticipate that our findings will facilitate in vitro gene expression systems that require membrane-dependent activities and open new opportunities in glycoengineering.

synthetic biology↗

bioRxiv2020

Cell-free prototyping of limonene biosynthesis using cell-free protein synthesis

Metabolic engineering of microorganisms to produce sustainable chemicals has emerged as an important part of the global bioeconomy. Unfortunately, efforts to design and engineer microbial cell factories are challenging because design-built-test cycles, iterations of re-engineering organisms to test and optimize new sets of enzymes, are slow. To alleviate this challenge, we demonstrate a cell-free approach termed in vitro Prototyping and Rapid Optimization of Biosynthetic Enzymes (or iPROBE). In iPROBE, a large number of pathway combinations can be rapidly built and optimized. The key idea is to use cell-free protein synthesis (CFPS) to manufacture pathway enzymes in separate reactions that are then mixed to modularly assemble multiple, distinct biosynthetic pathways. As a model, we apply our approach to the 9-step heterologous enzyme pathway to limonene in extracts from Escherichia coli. In iterative cycles of design, we studied the impact of 54 enzyme homologs, multiple enzyme levels, and cofactor concentrations on pathway performance. In total, we screened over 150 unique sets of enzymes in 580 unique pathway conditions to increase limonene production in 24 hours from 0.2 to 4.5 mM (23 to 610 mg/L). Finally, to demonstrate the modularity of this pathway, we also synthesized the biofuel precursors pinene and bisabolene. We anticipate that iPROBE will accelerate design-build-test cycles for metabolic engineering, enabling data-driven multiplexed cell-free methods for testing large combinations of biosynthetic enzymes to inform cellular design. TOC Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=50 SRC="FIGDIR/small/057737v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@19aa729org.highwire.dtl.DTLVardef@547698org.highwire.dtl.DTLVardef@13604b6org.highwire.dtl.DTLVardef@16f6718_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIApplied the iPROBE framework to build the nine-enzyme pathway to produce limonene C_LIO_LIAssessed the impact of cofactors and 54 enzyme homologs on cell-free enzyme performance C_LIO_LIIteratively optimized the cell-free production of limonene by exploring more than 580 unique reactions C_LIO_LIExtended pathway to biofuel precursors pinene and bisabolene C_LI

synthetic biology↗

bioRxiv2020

Cell-free protein synthesis enables one-pot cascade biotransformation in an aqueous-organic biphasic system

Biocatalytic cascade reactions have become increasingly important and useful for chemical synthesis. However, biocatalysts are often incompatible with organic solvents, which prohibits many cascade reactions involving nonpolar substrates. In this work, we used cell-free protein synthesis (CFPS) to express enzymes in an aqueous-organic biphasic system for the construction of an artificial enzymatic pathway. CFPS-expressed enzymes without purification performed efficiently to convert styrene (below 20 mM) to (S)-1-phenyl-1,2-ethanediol (two steps in one pot) with 100% conversion. In addition, our CFPS system showed great tolerance to different organic solvents and, importantly, the entire biocatalytic system can be consistently scaled up without reduction of the substrate conversion rate. We therefore anticipate that our cell-free approach will make possible cost-effective, high-yielding synthesis of valuable chemicals.

synthetic biology↗

bioRxiv2020

Development of a clostridia-based cell-free system for prototyping genetic parts and metabolic pathways

Gas fermentation by autotrophic bacteria, such as clostridia, offers a sustainable path to numerous bioproducts from a range of local, highly abundant, waste and low-cost feedstocks, such as industrial flue gases or syngas generated from biomass or municipal waste. Unfortunately, designing and engineering clostridia remains laborious and slow. The ability to prototype individual genetic parts, gene expression, and biosynthetic pathway performance in vitro before implementing them in cells could help address these bottlenecks by speeding up design. Unfortunately, a high-yielding cell-free gene expression (CFE) system from clostridia has yet to be developed. Here, we report the development and optimization of a high-yielding (236 {+/-} 24 {micro}g/mL) batch CFE platform from the industrially relevant anaerobe, Clostridium autoethanogenum. A key feature of the platform is that both circular and linear DNA templates can be applied directly to the CFE reaction to program protein synthesis. We demonstrate the ability to prototype gene expression, and quantitatively map cell-free metabolism in lysates from this system. We anticipate that the C. autoethanogenum CFE platform will not only expand the protein synthesis toolkit for synthetic biology, but also serve as a platform in expediting the screening and prototyping of gene regulatory elements in non-model, industrially relevant microbes.

synthetic biology↗

bioRxiv2020

Cell-free styrene biosynthesis at high titers

Styrene is an important petroleum-derived molecule that is polymerized to make versatile plastics, including disposable silverware and foamed packaging materials. Finding more sustainable methods, such as biosynthesis, for producing styrene is essential due to the increasing severity of climate change as well as the limited supply of fossil fuels. Recent metabolic engineering efforts have enabled the biological production of styrene in Escherichia coli, but styrene toxicity and volatility limit biosynthesis in cells. To address these limitations, we have developed a cell-free styrene biosynthesis platform. The cell-free system provides an open reaction environment without cell viability constraints, which allows exquisite control over reaction conditions and greater carbon flux toward product formation rather than cell growth. The two biosynthetic enzymes required for styrene production were generated via cell-free protein synthesis and mixed in defined ratios with supplemented L-phenylalanine and buffer. By altering the time, temperature, pH, and enzyme concentrations in the reaction, this approach increased the cell-free titer of styrene from 5.36 {+/-} 0.63 mM to 40.33 {+/-} 1.03 mM, an order of magnitude greater than cellular synthesis methods. Cell-free systems offer a complimentary approach to cellular synthesis of small molecules, which can provide particular benefits for producing toxic molecules. HighlightsA cell-free system for styrene biosynthesis was established. This in vitro system achieved styrene titers an order of magnitude greater than the highest reported concentration in vivo.

synthetic biology↗

bioRxiv2019

Apparent size and morphology of bacterial microcompartments varies with technique

Bacterial microcompartments (MCPs) are protein-based organelles which encapsulate metabolic pathways. Metabolic engineers have recently sought to repurpose MCPs to encapsulate heterologous pathways to increase flux through pathways of interest. As MCP engineering becomes more common, standardized methods for analyzing changes to MCPs and interpreting results across studies will become increasingly important. In this study, we demonstrate that different imaging techniques yield variations in the apparent size of purified MCPs from Salmonella enterica serovar Typhimurium LT2, likely due to variations in sample preparation methods. We provide guidelines for preparing samples for MCP imaging and outline expected variations in apparent size and morphology between methods. With this report we aim to establish an aid for comparing results across studies.

synthetic biology↗

bioRxiv2019

A cell-free biosynthesis platform for modular construction of protein glycosylation pathways

Glycosylation plays important roles in cellular function and endows protein therapeutics with beneficial properties. However, constructing biosynthetic pathways to study and engineer protein glycosylation remains a bottleneck. To address this limitation, we describe a modular, versatile cell-free platform for glycosylation pathway assembly by rapid in vitro mixing and expression (GlycoPRIME). In GlycoPRIME, crude cell lysates are enriched with glycosyltransferases by cell-free protein synthesis and then glycosylation pathways are assembled in a mix-and-match fashion to elaborate a single glucose priming handle installed by an N-linked glycosyltransferase. We demonstrate GlycoPRIME by constructing 37 putative protein glycosylation pathways, creating 23 unique glycan motifs. We then use selected pathways to design a one-pot cell-free system to synthesize a vaccine protein with an -galactose motif and engineered Escherichia coli strains to produce human antibody constant regions with minimal sialic acid motifs. We anticipate that our work will facilitate glycoscience and make possible new glycoengineering applications.

synthetic biology↗

bioRxiv2019

Design and optimization of a cell-free atrazine biosensor

Recent advances in cell-free synthetic biology have spurred the development of in vitro molecular diagnostics that serve as effective alternatives to whole-cell biosensors. However, cell-free sensors for detecting manmade organic water contaminants such as pesticides are sparse, partially because few characterized natural biological sensors can directly detect such pollutants. Here, we present a platform for the cell-free detection of one critical water contaminant, atrazine, by combining a previously characterized cyanuric acid biosensor with a reconstituted atrazine-to-cyanuric acid metabolic pathway composed of several protein-enriched bacterial extracts mixed in a one pot reaction. Our cell-free sensor detects atrazine within an hour of incubation at an activation ratio superior to previously reported whole-cell atrazine sensors. We also show that the response characteristics of the atrazine sensor can be tuned by manipulating the component ratios of the cell-free reaction mixture. Our approach of utilizing multiple metabolic steps, encoded in protein-enriched cell-free extracts, to convert a target of interest into a molecule that can be sensed by a transcription factor is modularly designed, which should enable this work to serve as an effective proof-of-concept for rapid field-deployable detection of complex organic water contaminants.

synthetic biology↗

bioRxiv2019

Design of a transcriptional biosensor for the portable, on-demand detection of cyanuric acid

Rapid molecular biosensing is an emerging application area for synthetic biology. Here, we engineer a portable biosensor for cyanuric acid (CYA), an analyte of interest for human and environmental health, using a LysR-type transcription regulator (LTTR) from Pseudomonas within the context of Escherichia coli gene expression machinery. To overcome cross-host portability challenges of LTTRs, we rationally engineered hybrid Pseudomonas-E. coli promoters by integrating DNA elements required for transcriptional activity and ligand-dependent regulation from both hosts, which enabled E. coli to function as whole-cell biosensor for CYA. To alleviate challenges of whole-cell biosensing, we adapted these promoter designs to function within a freeze-dried E. coli cell-free system to sense CYA. This portable, on-demand system robustly detects CYA within an hour from laboratory and real-world samples and works with both fluorescent and colorimetric reporters. This work elucidates general principles to facilitate the engineering of a wider array of LTTR-based environmental sensors.

synthetic biology↗

bioRxiv2019

Point-of-Use Detection of Environmental Fluoride via a Cell-Free Riboswitch-Based Biosensor

Advances in biosensor engineering have enabled the design of programmable molecular systems to detect a range of pathogens, nucleic acids, and chemicals. Here, we engineer and field-test a biosensor for fluoride, a major groundwater contaminant of global concern. The sensor consists of a cell-free system containing a DNA template that encodes a fluoride-responsive riboswitch regulating genes that produce a fluorescent or colorimetric output. Individual reactions can be lyophilized for long-term storage and detect fluoride at levels above 2 parts per million, the EPAs most stringent regulatory standard, in both laboratory and field conditions. Through onsite detection of fluoride in a real-world water source, this work provides a critical proof-of-principle for the future engineering of riboswitches and other biosensors to address challenges for global health and the environment.

synthetic biology↗

bioRxiv2019

In vitro ribosome synthesis and evolution through ribosome display

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. However, our recent development of an integrated strategy for the in vitro synthesis and assembly of translationally competent ribosomes (iSAT) enables the rapid generation of large libraries of ribosome variants in a cell-free environment. Here we combine the iSAT system with ribosome display to develop a fully in vitro methodology for ribosome synthesis and evolution (called RISE). We validate this method by selecting highly active genotypes which are resistant to the antibiotic clindamycin from a library of ribosome variants. We further demonstrate the prevalence of positive epistasis in successful 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.

synthetic biology↗

bioRxiv2019

In vitro prototyping and rapid optimization of biosynthetic enzymes for cellular design

Microbial cell factories offer an attractive approach for production of biobased products. Unfortunately, designing, building, and optimizing biosynthetic pathways remains a complex challenge, especially for industrially-relevant, non-model organisms. To address this challenge, we describe a platform for in vitro Prototyping and Rapid Optimization of Biosynthetic Enzymes (iPROBE). 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 with two examples. First, we tested and ranked 54 different pathways for 3-hydroxybutyrate production, improving in vivo production in Clostridium by 20-fold to 14.63 {+/-} 0.48 g/L and identifying a new biosynthetic route to (S)-(+)-1,3-butanediol. Second, we used iPROBE and data-driven design to optimize a 6-step n-butanol pathway, increasing titers 4-fold across 205 pathways, and showed strong correlation between cell-free and cellular performance. We expect iPROBE to accelerate design-build-test cycles for industrial biotechnology.

synthetic biology↗

bioRxiv2019

On-demand, cell-free biomanufacturing of conjugate vaccines at the point-of-care

Conjugate vaccines are among the most effective methods for preventing bacterial infections, representing a promising strategy to combat drug-resistant pathogens. 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 bioconjugate vaccine expression (iVAX) in portable, freeze-dried lysates from detoxified, nonpathogenic Escherichia coli. Upon rehydration, iVAX reactions synthesize clinically relevant doses of bioconjugate vaccines against diverse bacterial pathogens in one hour. We show that iVAX synthesized vaccines against the highly virulent pathogen Franciscella tularensis subsp. tularensis (type A) strain Schu S4 elicited pathogen-specific antibodies in mice at significantly higher levels compared to vaccines produced using engineered bacteria. The iVAX platform promises to accelerate development of new bioconjugate vaccines with increased access through refrigeration-independent distribution and point-of-care production.

synthetic biology↗

bioRxiv2018

Deconstructing cell-free extract preparation for in vitro activation of transcriptional genetic circuitry

Recent advances in cell-free gene expression (CFE) systems have enabled their use for a host of synthetic biology applications, particularly for rapid prototyping of genetic circuits designed as biosensors. Despite the proliferation of cell-free protein synthesis platforms, the large number of currently existing protocols for making CFE extracts muddles the collective understanding of how the method by which an extract is prepared affects its functionality. Specifically, a key goal toward developing cell-free biosensors based on native genetic regulators is activating the transcriptional machinery present in bacterial extracts for protein synthesis. However, protein yields from genes transcribed in vitro by the native Escherichia coli RNA polymerase are quite low in conventional crude extracts originally optimized for expression by the bacteriophage transcriptional machinery. Here, we show that cell-free expression of genes under bacterial {sigma}70 promoters is constrained by the rate of transcription in crude extracts and that processing the extract with a ribosomal run-off reaction and subsequent dialysis can alleviate this constraint. Surprisingly, these processing steps only enhance protein synthesis in genes under native regulation, indicating that the translation rate is unaffected. We further investigate the role of other common process variants on extract performance and demonstrate that bacterial transcription is inhibited by including glucose in the growth culture, but is unaffected by flash-freezing the cell pellet prior to lysis. Our final streamlined protocol for preparing extract by sonication generates extract that facilitates expression from a diverse set of sensing modalities including protein and RNA regulators. We anticipate that this work will clarify the methodology for generating CFE extracts that are active for biosensing and will encourage the further proliferation of cell-free gene expression technology for new applications.

synthetic biology↗

bioRxiv2018

Selection removes Shine-Dalgarno-like sequences from within protein coding genes

The Shine-Dalgarno (SD) sequence motif facilitates translation initiation and is frequently found upstream of bacterial start codons. However, thousands of instances of this motif occur throughout the middle of protein coding genes in a typical bacterial genome. Here, we use comparative evolutionary analysis to test whether SD sequences located within genes are functionally constrained. We measure the conservation of SD sequences across Gammaproteobacteria, and find that they are significantly less conserved than expected. Further, the strongest SD sequences are the least conserved whereas we find evidence of conservation for the weakest possible SD sequences given amino acid constraints. Our findings indicate that most SD sequences within genes are likely to be deleterious and removed via selection. To illustrate the origin of these deleterious costs, we show that ATG start codons are significantly depleted downstream of SD sequences within genes, highlighting the potential for these sequences to promote erroneous translation initiation.

evolutionary biology↗

bioRxiv2017

Computational Design of Asymmetric Three-dimensional RNA Structures and Machines.

The emerging field of RNA nanotechnology seeks to create nanoscale 3D machines by repurposing natural RNA modules, but successes have been limited to symmetric assemblies of single repeating motifs. We present RNAMake, a suite that automates design of RNA molecules with complex 3D folds. We first challenged RNAMake with the paradigmatic problem of aligning a tetraloop and sequence-distal receptor, previously only solved via symmetry. Single-nucleotide-resolution chemical mapping, native gel electrophoresis, and solution x-ray scattering confirmed that 11 of the 16 miniTTR designs successfully achieved clothespin-like folds. A 2.55 [A] diffraction-resolution crystal structure of one design verified formation of the target asymmetric nanostructure, with large sections achieving near-atomic accuracy (< 2.0 [A]). Finally, RNAMake designed asymmetric segments to tether the 16S and 23S rRNAs together into a synthetic singlestranded ribosome that remains uncleaved by ribonucleases and supports life in Escherichia coli, a challenge previously requiring several rounds of trial-and-error.

bioengineering↗