Bottleneck/Challenge: Inability to produce cell-free systems in a scalable and standardized fashion, protocols for cell-free systems generation vary from lab-to-lab, and reactions are carried out at very small volumes (1-10µl) that limit the extent to which we can characterize the systems; for example, we lack an understanding of how the metabolic state of an extract impacts energy usage, protein synthesis, and DNA/RNA regulation.

Potential Solution: Arrive at a standardized bacterial cell-free system extract generation and reaction protocol that is robust, inexpensive, and would allow their routine scalable production; this includes identification of energy mixtures robust to various extract preparation procedures.

Potential Solution: Use -omics tools (e.g., mass spectrometry, next generation sequencing) to understand batch-to-batch and lab-to-lab lysate quality and composition variability.

Potential Solution: Define an equipment set for bacterial lysate preparation (i.e., lysis technique).

Bottleneck/Challenge: Ad hoc use of different extract preparation procedures, cell-free reaction conditions, and reporter construct architectures lead to challenges in making reproducible and comparable cell-free systems.

Potential Solution: Design cell-free system physiochemical composition that is robust to different process parameters, or identify compositions for each defined extract preparation method.

Potential Solution: Identify and implement measurement techniques needed to facilitate reproducibility (e.g., standard assay for quantifying activity across labs).

Potential Solution: Rigorously characterize and understand how reaction geometries and surface area-to-volume impact cell-free system performance.

Potential Solution: Create a plasmid repository specific to bacterial cell-free systems (including T7 constructs and E. coli promoters), with a description of performance (such as protein expression kinetics).

Potential Solution: Develop a quantitative ‘culture’ to cell-free system practices.

Bottleneck/Challenge: Gaps in understanding critical components and how they vary across species and kingdoms (e.g., prokaryotes vs. eukaryotes).

Potential Solution: Assess critical parameters with design-of-experiments and machine learning across strains and procedures.

Potential Solution: Develop an online tool to customize the components of a cell-free reaction for a given organism and approximate RNA/protein output, reveal components that should have been included, and application-specific assembly (metabolic engineering vs. protein synthesis vs. circuits).

Bottleneck/Challenge: Ability to regenerate energy and cofactors with native metabolism is constrained in non-E. coli platforms.

Potential Solution: Assess metabolic pathways that could be activated to facilitate energy regeneration.

Potential Solution: Develop exogenous cofactor regeneration modules to drop in and out when native ones are missing.

Potential Solution: Incorporate genome-scale models to predict energy and cofactor regeneration systems most suitable for a new host.

Bottleneck/Challenge: Gaps in understanding critical components of host lysates that make each unique and which to use for a specific, desired product.

Potential Solution: Identify the critical species-specific components (using -omics approaches) and experimentally validate these components (including transcription factors, accessory proteins for translation, and metabolic modules).

Bottleneck/Challenge: Production of lysate relies on scaled-up culturing of sometimes recalcitrant cells.

Potential Solution: Understand which extract preparation protocol parameters should/can be tuned to match a new organism (i.e., trace the physiology of an organism to the extract preparation parameters).

Bottleneck/Challenge: Identify units of operation required for the scale-up and scale-out production of cell-free systems.

Potential Solution: Identify quality assurance and quality control metrics at each unit of operation that would allow the scale-up of the process.

Bottleneck/Challenge: Multiple minimal genomes necessary for building a cell have been proposed but none have yet been demonstrated to work in this context.

Potential Solution:: Define a set of possible minimal genomes that could enable self-replication of a minimal cell and make these component parts.

Potential Solution: Test each component of a minimal cell individually (including metabolic modules, regulation by most types of regulators, etc.).

Bottleneck/Challenge: We do not yet know how to build genomes de novo, and native genomes have built-in regulation that may not be suitable for engineered biological systems.

Potential Solution: Create and refactor modularized pathways for building a minimal cell.

Potential Solution: Show examples of a synthetic self-regulating gene cluster.

Potential Solution: Exploit CRISPR as a potential universal regulation mechanism to regulate synthetic minimal genomes.

Bottleneck/Challenge: Typical cell-free reactions that serve as the basis for minimal cells use an energy-rich environment of nucleotide triphosphates and high energy phosphate bond donors for chemical energy; these systems are short-lived, expensive, and can lead to inhibitory byproducts.

Potential Solution: Create generalized approaches for cost-effective, long-lived energy regeneration modules that can integrate with synthetic cells, which may require compartmentalized systems.

Potential Solution: Integrate efficient physical ATP regeneration systems in synthetic cells, such as photosynthesis.

Bottleneck/Challenge: Constructing ribosomes built completely of in vitro synthesized parts has remained elusive.

Potential Solution: Develop conditions that facilitate co-synthesis of all ribosome proteins and ribosomal RNA, first in extracts, then in purified systems.

Bottleneck/Challenge: The palette of biological chemistry is smaller than chemistry.

Potential Solution: Build hybrid biological-chemical systems.

Potential Solution: Expand the chemistry of genetically-encoded systems.

For related reading, please see Host Engineering Breakthrough Capability: Spatial control over (or organization of) metabolic pathways in cells and construction of unnatural organelles.

Bottleneck/Challenge: Lack a precise physical understanding of how the composition of the compartment influences encapsulation, transport, and retention of various types of cargo.

Potential Solution: Quantify the relationship between compartment composition and encapsulation efficiency, permeability, and stability as a function of compartment cargo.

Bottleneck/Challenge: Communication strategies for synthetic cells are not yet well-developed.

Potential Solution: Identify and implement mechanisms for communication between engineered compartments that allows efficient, reliable, multi-channel signaling between synthetic cells.

Potential Solution: Identify complementary signaling modules with reliable performance in a cell-free environment.

Bottleneck/Challenge: Inability to generate stable liposomes with the correct size and membrane-permeability to generate micro-vesicles (synthetic cells) with different chemical environments.

Potential Solution: Use microfluidics for precise production of chemically-defined vesicles.

Potential Solution: Exploit non-natural compartments, such as block copolymers, as a means to make mechanically robust compartments with negligible non-specific permeability.

Bottleneck/Challenge: Stability of reagents is not well understood in non-E. coli-based cell-free systems.

Potential Solution: The full spectrum of metabolic activity during cell-free reactions over time can be characterized in multiple systems, including those emerging cell-free systems, to identify reagent instabilities.

Bottleneck/Challenge: High-energy phosphate compounds are still commonly used to fuel cell-free systems (e.g., PEP, 3PGA); these compounds are expensive, lead to inhibitory phosphate concentrations, and only enable bursts of ATP regeneration instead of long-lived energy regeneration.

Potential Solution: Central metabolism, non-phosphorylated energy substrates, or light driven approaches (e.g., from bacteriorhodopsin), among others, could be used to enable long-lived energy regeneration without inhibitory byproducts; should be tested in extracts made from organism across all kingdoms and identify key factors (such as, concentrations, additional reagents, etc.) for robust alternative energy activation.

Bottleneck/Challenge: Cofactor regeneration and balancing strategies preclude long-term activation of energy metabolism to fuel metabolic activity.

Potential Solution: Molecular purge valves implemented in crude extracts could maintain redox balance.

Potential Solution: Develop schemes for balancing ATP, and derivatives including NAD(P) and FAD.

Potential Solution: Create non-natural cofactors, and requisite engineered enzymes, that have better stability properties.

Bottleneck/Challenge: Reagent instabilities limit reaction time.

Potential Solution: Genomic modifications to extract source strains can be carried out to stabilize substrates.

Bottleneck/Challenge: Small molecule byproducts of metabolism, such as phosphate, inhibit cell-free reactions.

Potential Solution: Engineer systems to avoid the accumulation of chemical inhibitors.

Potential Solution: Develop product siphoning strategies and dialysis-like strategies to remove inhibitors from the system.

Potential Solution: Engineer molecular complexes that are resistant to byproducts and chemical products.

Bottleneck/Challenge: Cell-free reactions terminate because substrates and cofactors are depleted, byproducts accumulate to inhibit the reaction, or the catalysts become inactivated; of these, enzyme stability represents a significant technical and economic hurdle to technology development in this space.

Potential Solution: Characterize catalyst instabilities such as, tolerance to byproducts and inhibitors.

Potential Solution: Increase stability of enzymes involved in cell-free systems.

Potential Solution: Generate design criteria for choosing catalysts (i.e., identifying select organisms from which to derive lysates and enzymes) for desired applications.

Potential Solution: Develop reactor designs and bioprocesses that continuously replenish the source of catalysts.

Bottleneck/Challenge: Without enzymes or lysates that are stable on the order of weeks, significant fractions of carbon will otherwise be used in generating the biocatalysts required of these systems.

Potential Solution: Integrate knowledge of the system and innovations to facilitate long-lived reactions.

Bottleneck/Challenge: Sequence-function data are limited by colorimetric and fluorescent read-outs.

Potential Solution: Develop next-generation, deep-sequencing-based approaches for monitoring transcription and translation in cell-free reactions, including transcription factor metabolite interactions.

Bottleneck/Challenge: There are limited numbers of sufficiently-large datasets available that allow comparison of genetic part performance and the development of modeling frameworks between the cell-free environment and in vivo.

Potential Solution: Develop cell-free systems for 20 industrially-relevant organisms that could form a testbed to establish libraries of new genetic parts and how to accelerate design.

Potential Solution: Develop a repository of genetic parts for cell-free systems, including performances.

Potential Solution: Develop a quantitative modeling platform specific to cell-free systems that takes into account the advantages and limitations of cell-free expression.

Bottleneck/Challenge: Resource limitations in cell-free systems (e.g., energy and cofactor regeneration) constrain the construction of multi-gene systems encoded by complex genetics, as well as time-dynamics needed to assess their function.

Potential Solution: Create robust, long-lived cell-free systems that can be routinely used for activating and characterizing multi-gene circuits.

Bottleneck/Challenge: There are limited numbers of sufficiently-large datasets available that allow comparison of genetic circuit performance and the development of modeling frameworks between the cell-free environment and in vivo.

Potential Solution: Develop cell-free systems for 20 industrially-relevant organisms which could form a testbed to establish libraries of new genetic circuits.

Bottleneck/Challenge: Lack of knowledge of how to activate essential components in eukaryotic cell-free systems.

Potential Solution: Make more lysates from eukaryotes and eukaryotic-like systems to be able to assess essential components.

Bottleneck/Challenge: Transcription and translation are typically combined in cell-free systems requiring viral sequences (e.g., internal ribosome entry site) for translation initiation rather than a 5’cap and polyA tail.

Potential Solution: Develop strategies to compartmentalize transcription to better mimic the natural process.

Bottleneck/Challenge: Sufficient transcription and translation activity is necessary to assess genetic designs and metabolic pathways.

Potential Solution: Streamline and provide generalized approaches to enable sufficient cell-free activity for gene expression and biosynthesis from diverse species.

Potential Solution: Use mass spectrometry to determine the proteome composition and metabolite composition of non-model organisms to accelerate the optimization of cell-free systems.

Bottleneck/Challenge: Expand cell-free systems to poorly explored ares, such as extremophiles, for engineering biology far from standard conditions.

Potential Solution: Select a set of extremophiles that can be grown in laboratories and easily lysed.

Bottleneck/Challenge: Toxicity of cell-free components is not well understood.

Potential Solution: Evaluate toxicity of cell-free components.

Bottleneck/Challenge: Lipopolysaccharides, also known as lipoglycans and endotoxins, are large molecules consisting of a lipid and a polysaccharide composed of O-antigen which can cause toxicity of products manufactured using bacteria.

Potential Solution: Cost-effective strategies to remove endotoxin is required to ensure safety.

Potential Solution: Create bacterial species that are detoxified by design.

Bottleneck/Challenge: A major limitation of traditional sensors and centralized medicine manufacturing is that the products must be refrigerated; cell-free systems can be freeze-dried for potential room-temperature storage and distribution, but suffer from activity loss in certain conditions.

Potential Solution: Create stable, freeze-dried systems for storage without a cold-chain by using cryoprotectants and process modifications.

Potential Solution: Demonstrate multiple reaction formats (i.e., pellets, gels, paper, etc.) stable for 2-5 years.

Bottleneck/Challenge: Manufacturing medicines in rapid response to emerging and re-emerging threats requires fast protein synthesis rates and reactions that can be completed in minutes to hours.

Potential Solution: For translation outputs, increase the overall catalyst concentration in the reaction to enhance reaction rates.

Potential Solution: When possible, develop transcriptional outputs (such as for sensors) which offer a significant speed improvement (minutes versus hours).

Bottleneck/Challenge: Completely automated operation platform to manufacture and purify proteins suitable for the FDA has not yet been fully validated.

Potential Solution: Build integrated units and measure the repeatability (of the same unit) and reproducibility (between units) of the system.

Bottleneck/Challenge: Point-of-care synthesis and administration of glycoprotein therapeutics and vaccines requires robust production and purification methods.

Potential Solution: Develop simple, portable purification systems which can reliably produce FDA-compliant vaccines and therapeutics from cell-free production systems and demonstrate efficiency in animal models.

Bottleneck/Challenge: Many of the most important components of glycosylation pathways are associated with cellular membranes and cannot be recapitulated easily in cell-free systems.

Potential Solution: Develop and optimize efficient strategies use of oligosaccharyltransferases (including eukaryotic versions) to transfer pre-built sugars from lipid-linked oligosaccharides in vitro.

Potential Solution: Develop methods to select and assemble a set of soluble enzymatic tools capable of producing therapeutically-relevant glycoproteins with desired properties in vitro from simple, commercially available activated-sugar building blocks.

Bottleneck/Challenge: Synthesis of complex human glycans in cell-free systems is constrained by the available set of well characterized enzymes; existing characterizations do not provide sufficient tools to go from a set of sugar monomers and a design to a glycoprotein of interest.

Potential Solution: Expand the glycoengineering toolkit by characterizing glycoslytransferases to assemble sets of well-characterized enzymes that can reliably produce desired glycoproteins.

Potential Solution: Engineer existing glycosylation enzymes for desired activities when naturally occurring enzymes with desired functionalities are not available (e.g., engineering of the bacterial oligosaccharyltransferase to accept the eukaryotic core glycan would enable the efficient production of the eukaryotic core glycan in bacterial systems).

Bottleneck/Challenge: The production of glycoconjugate vaccines against bacteria require the culturing of pathogenic strains recalcitrant to bioengineering and the use of non-specific conjugation chemistry.

Potential Solution: Express diverse bacterial O-antigen pathways for characterization and production of lipid-linked oligosaccharides required for vaccines and use bacterial oligosaccharyltransferases to site-specifically attach these oligosaccharides in vitro.

Potential Solution: Use the greater control afforded by cell-free protein synthesis systems to produce FDA-approved vaccine carrier and antigen proteins, many of which cannot be produced outside of pathogenic organisms.

Bottleneck/Challenge: Glycosylation of therapeutically-relevant metabolites (including many antibiotics) is often required for desirable pharmacokinetic/dynamic properties, but many of the enzymes which perform these glycosylation activities are unknown or can only be expressed in their native host strain.

Potential Solution: Use cell-free protein synthesis to screen the activity of metabolite-targeting glycosyltransferases on existing bioactive compound libraries to understand their specificities and improve the therapeutic utility of small molecule products.

Bottleneck/Challenge: Substrate limitations lead to inherent inefficiencies.

Potential Solution: Develop metabolic models of lysate hosts to identify genetic knockouts that enhance glycan production and carry out such modifications.

Bottleneck/Challenge: Development timelines of glycoprotein therapeutics are slowed by the need to produce these products in mammalian cell lines.

Potential Solution: Develop, optimize, and implement strategies to create more than ten unique and homogeneous glycan structures on proteins by cell-free methods (e.g., trimannose core eukaryotic glycan, afucosylated galactose-terminated core glycan, fucosylated sialic-acid terminated core glycan, biantennary glycan, etc.); once synthesized, these products can studied and optimized using therapeutic functionality assays.

Bottleneck/Challenge: Intentional engineering of glycan structures and the synthesis of novel structures is constrained by identifying sets of enzymes for the manufacture of the sugar structures.

Potential Solution: Develop cell-free systems capable of rapidly and robustly producing any defined glycoprotein on-demand.