Protocol for preparation of cell-free system.

Cell-free protein synthesis (CFPS) has emerged as a powerful tool for protein production, with applications ranging from basic research to biotechnology and pharmaceutical development. However, enhancing the efficiency of CFPS systems remains a crucial challenge for realizing their full potential. Computational strategies offer promising avenues for optimizing CFPS efficiency by providing insights into complex biological processes and enabling rational design approaches. This review provides a comprehensive overview of the computational approaches aimed at enhancing CFPS efficiency. The introduction outlines the significance of CFPS and the role of computational methods in addressing efficiency limitations. It discusses mathematical modeling and simulation-based approaches for predicting protein synthesis kinetics and optimizing CFPS reactions. The review also delves into the design of DNA templates, including codon optimization strategies and mRNA secondary structure prediction tools, to improve protein synthesis efficiency. Furthermore, it explores computational techniques for engineering cell-free transcription and translation machinery, such as the rational design of expression systems and the predictive modeling of ribosome dynamics. The predictive modeling of metabolic pathways and the energy utilization in CFPS systems is also discussed, highlighting metabolic flux analysis and resource allocation strategies. Machine learning and artificial intelligence approaches are being increasingly employed for CFPS optimization, including neural network models, deep learning algorithms, and reinforcement learning for adaptive control. This review presents case studies showcasing successful CFPS optimization using computational methods and discusses applications in synthetic biology, biotechnology, and pharmaceuticals. The challenges and limitations of current computational approaches are addressed, along with future perspectives and emerging trends, such as the integration of multi-omics data and advances in high-throughput screening. The conclusion summarizes key findings, discusses implications for future research directions and applications, and emphasizes opportunities for interdisciplinary collaboration. This review offers valuable insights and prospects regarding computational strategies to enhance CFPS efficiency. It serves as a comprehensive resource, consolidating current knowledge in the field and guiding further advancements.

A new wave of interest in cell-free protein synthesis (CFPS) systems has shown their utility for producing proteins at high titers, establishing genetic regulatory element libraries ( e.g., promoters, ribosome binding sites) in nonmodel organisms, optimizing biosynthetic pathways before implementation in cells, and sensing biomarkers for diagnostic applications. Unfortunately, most previous efforts have focused on a select few model systems, such as Escherichia coli. Broadening the spectrum of organisms used for CFPS promises to better mimic host cell processes in prototyping applications and open up new areas of research. Here, we describe the development and characterization of a facile CFPS platform based on lysates derived from the fast-growing bacterium Vibrio natriegens, which is an emerging host organism for biotechnology. We demonstrate robust preparation of highly active extracts using sonication, without specialized and costly equipment. After optimizing the extract preparation procedure and cell-free reaction conditions, we show synthesis of 1.6 ± 0.05 g/L of superfolder green fluorescent protein in batch mode CFPS, making it competitive with existing E.coli CFPS platforms. To showcase the flexibility of the system, we demonstrate that it can be lyophilized and retain biosynthesis capability, that it is capable of producing antimicrobial peptides, and that our extract preparation procedure can be coupled with the recently described Vmax Express strain in a one-pot system. Finally, to further increase system productivity, we explore a knockout library in which putative negative effectors of CFPS are genetically removed from the source strain. Our V.natriegens-derived CFPS platform is versatile and simple to prepare and use. We expect it will facilitate expansion of CFPS systems into new laboratories and fields for compelling applications in synthetic biology.

Cell-free systems are emerging as powerful platforms for synthetic biology with widespread applications in both fundamental research, such as artificial cell construction, and practical uses like recombinant protein production. Among these, cell-free protein synthesis (CFPS) plays a crucial role in gene expression for various downstream applications. However, the development of CFPS systems based on certain chassis, such as Bacillus subtilis, still remains limited due to their low in vitro productivity. Here, we report the development of a highly productive CFPS system derived from an engineered B. subtilis 164T7P strain, which contains a genomic integration of the T7 RNA polymerase gene. This modification allows the preparation of cell extracts that inherently contain T7 RNA polymerase, enabling T7 promoter-based transcription without the supplementation of purified T7 RNA polymerase in CFPS reactions. Through systematic optimization of cell extract preparation and key reaction parameters, we achieved the synthesis of 286 ± 16.7 μg/mL of sfGFP in batch reactions, with yields increasing to over 1100 μg/mL in a semicontinuous format that can replenish substrates and remove inhibitory byproducts. We further demonstrated the system's versatility by using it for two synthetic biology applications: prototyping ribosome binding site (RBS) elements and synthesizing pulcherriminic acid─a bioactive cyclodipeptide. The system successfully characterized RBS performance, with in vitro and in vivo rankings correlating with predicted strengths, and expressed two active biosynthetic enzymes (cyclodipeptide synthase─YvmC and cytochrome P450 enzyme─CypX), leading to the production of pulcherriminic acid. Overall, our B. subtilis-based CFPS system offers a robust platform for high-yield protein synthesis, in vitro prototyping of gene regulatory elements, and natural product biosynthesis, highlighting its broad potential for synthetic biology and biotechnology applications.

Virus-like particles (VLPs) are supramolecular protein assemblies with the potential for unique and exciting applications in synthetic biology and medicine. Despite the attention VLPs have gained thus far, considerable limitations still persist in their production. Poorly scalable manufacturing technologies and inconsistent product architectures continue to restrict the full potential of VLPs. Cell-free protein synthesis (CFPS) offers an alternative approach to VLP production and has already proven to be successful, albeit using extracts from a limited number of organisms. Using a recently developed Pichia pastoris-based CFPS system, we have demonstrated the production of the model Hepatitis B core antigen VLP as a proof-of-concept. The VLPs produced in the CFPS system were found to have comparable characteristics to those previously produced in vivo and in vitro. Additionally, we have developed a facile and rapid synthesis, assembly and purification methodology that could be applied as a rapid prototyping platform for vaccine development or synthetic biology applications. Overall the CFPS methodology allows far greater throughput, which will expedite the screening of optimal assembly conditions for more robust and stable VLPs. This approach could therefore support the characterization of larger sample sets to improve vaccine development efficiency.

Expanding the palette of Streptomyces-based cell-free protein synthesis systems with enhanced yields

Chapter 24 - Cell-free synthetic biology as an emerging biotechnology

Cell‐like platforms are being studied intensively for their application in synthetic biology to mimic aspects of life in an artificial environment. Here, micrometer‐sized, bifunctional microgels are used as an experimental platform to investigate the interplay of cell‐free protein synthesis (CFPS) and in situ protein accumulation inside the microgel volume. In detail, microgels made of hyaluronic acid (HA) are first modified with different amounts of nitrilotriacetic acid (NTA) moieties to characterize the capability and maximum capacity of binding His‐tag modified GFP. CFPS is optimized for the system used here, particularly when using a linear DNA template. Afterward, HA‐microgels are functionalized with the linear DNA template and Ni2+‐activated NTA moieties to bind in situ synthesized GFP‐His. CFPS and parallel protein accumulation within the microgels are observed over time to determine the GFP‐His binding to the microgel platform. With this approach, the study presents the first steps for a platform to study the temporal‐spatial regulation of protein synthesis by tailored protein binding or release from the microgel matrix‐based reaction environment.

Synthetic biology is an emerging interdisciplinary field covering the subjects of biology, chemistry and engineering, which promotes the progress from understanding life to creating living systems. As one of important forefront directions of synthetic biology, in vitro synthetic biology (IVSB) focuses on studying the phenomenon and rules of life activities in testing tubes, not relying on living organisms. Cell-free protein synthesis (CFPS) is an effective research platform for IVSB, which utilizes exogenous DNA or mRNA as genetic information for synthesizing protein in vitro , instead of traditional protein expression in cells. CFPS possesses superior advantages over cell-based systems including convenience, simplicity, rapidness, excellent controllability and green economy. Moreover, CFPS is a robust research model for exploration of life evolution and creation of artificial living system. Since 1958, it has been demonstrated for the first time that proteins could be synthesized from cell extract; and afterwards, CFPS has emerged to not only a promising platform for proteomics and pharmaceutics, but also a significant tool for revelation of chemical basis of cells and exploration of origins of life. Cell extracts contained basic transcription and translation machineries, energy regeneration substrates, amino acids, nucleotides and cofactors. A variety of cell extracts have been created from different cell sources such as Escherichia coli ( E.coli ), wheat germ, yeast and rabbit reticulocyte. E.coli was the earliest prokaryotic system and most widely used due to its high production yield, short production cycle and low cost. The protein yields of E. coli system could reach up to several milligrams per milliliter of reaction. Eukaryotic systems such as wheat germ and rabbit reticulocyte although less productive, provided more robust platform for studying protein functions, in particular for post-translationally modified proteins. Different production formats of CFPS have been developed and classified according to how the reactions were fed, including batch, continuous-flow, continuous-exchange, bilayer and hydrogel. Batch was the simplest format, but the key limitation was the short lifetimes and the consequent low yield. This was primarily because of the rapid depletion of the high-energy phosphate pool, leading to accumulation of free phosphates and further inhibition of protein synthesis. Continuous-flow overcame the problem in batch format because of the continuous supply of energy and substrates and the continuous removal of the by-products. As a result, the reaction time could be extended to 20 hours, and the product yield was increased by two orders of magnitude in comparison with batch format. In continuous-exchange format, passive exchange of substrates and by-products further extended the reaction lifetime. However, continuous formats were not easily applicable to high-throughput processes, in which miniaturization and automation were required. Bilayer format was simple, efficient and high-throughput-friendly. Very recently, hydrogel formats have been developed because of the gel niche resembling the physicochemical nature of cells, subsequently leading to high yield. One of the promising applications of CFPS was to synthesize biologically active proteins. CFPS could produce proteins that were difficult or impossible to express with cell-based systems, such as membrane proteins, cell-toxic proteins, isotope-labeling proteins and proteins with unnatural amino acids incorporated. The open nature and high versatility of the CFPS platform enabled high-throughput tools for genomics and proteomics, allowing for biopharmaceutical applications. Through the high- throughput screening of proteins in CFPS the efficacy of protein drugs could be significantly improved by addition of unnatural amino acids, which provided excellent pharmacological properties and prolonged the half-life of drugs in plasma. In this review, we therefore summarize the development history, compositions, categories and production formats of CFPS, as well as applications in the synthesis of functional proteins, high throughput synthesis and screening of protein, and production of protein drugs.

Cell-free Protein Synthesis, or (CFPS), is an interesting innovation in synthetic biology that allows for precise and controlled manufacturing of proteins and biomolecules. This review explores the progress made in CFPS techniques, especially in lysate preparation, computational modelling, gene impairments, and system improvements. Advances in optimized ribosomes, synthetic genetic circuits, and codon usage have improved how big and how well CFPS systems can work. Also, the combination of microfluidics, new energy supply systems, and linear DNA templates has broadened the field of application of CFPS in bioprocess, targeted therapies and ecotoxicological assessment. However, other problems such as the depletion of resources, accumulation of byproducts and protease activity have ranked higher than these advancements, which calls for updating of the system and control of the process. This review also discusses the mRNA secondary structure elements, translation regulation elements and translation enhancing tools that have been employed to increase the efficiency of translation. The predicted growth of CFPS is predicated on its capacity to expand, incorporate biotechnological advancements and break biochemical barriers, hence making it a critical technology in the next generation of therapeutic as well as diagnostic interventions.

Cell-free protein synthesis: Recent advances in bacterial extract sources and expanded applications

Cell-free protein synthesis (CFPS) technologies have enabled inexpensive and rapid recombinant protein expression. Numerous highly active CFPS platforms are now available and have recently been used for synthetic biology applications. In this review, we focus on the ability of CFPS to expand our understanding of biological systems and its applications in the synthetic biology field. First, we outline a variety of CFPS platforms that provide alternative and complementary methods for expressing proteins from different organisms, compared with in vivo approaches. Next, we review the types of proteins, protein complexes, and protein modifications that have been achieved using CFPS systems. Finally, we introduce recent work on genetic networks in cell-free systems and the use of cell-free systems for rapid prototyping of in vivo networks. Given the flexibility of cell-free systems, CFPS holds promise to be a powerful tool for synthetic biology as well as a protein production technology in years to come.

Synthetic biology is built on the synthesis, engineering, and assembly of biological parts. Proteins are the first components considered for the construction of systems with designed biological functions because proteins carry out most of the biological functions and chemical reactions inside cells. Protein synthesis is considered to comprise the most basic levels of the hierarchical structure of synthetic biology. Cell-free protein synthesis has emerged as a powerful technology that can potentially transform the concept of bioprocesses. With the ability to harness the synthetic power of biology without many of the constraints of cell-based systems, cell-free protein synthesis enables the rapid creation of protein molecules from diverse sources of genetic information. Cell-free protein synthesis is virtually free from the intrinsic constraints of cell-based methods and offers greater flexibility in system design and manipulability of biological synthetic machinery. Among its potential applications, cell-free protein synthesis can be combined with various man-made devices for rapid functional analysis of genomic sequences. This review covers recent efforts to integrate cell-free protein synthesis with various reaction devices and analytical platforms.

This work draws inspiration from totipotent cellular systems to design smart materials whose compositions and properties can be learned or evolved. Totipotency refers to the inherent genetic potential of a single cell to adapt and produce all types of differentiated cells within an organism. To study this principal and apply it synthetically, tissue-like compartmentalized assemblies are constructed via lipid membrane-separated aqueous droplets in a hydrophobic medium through the droplet interface bilayer (DIB) method. Within our droplets, we explore synthetic totipotency via cell-free reactions including actin polymerization and cell free protein synthesis (CFPS). The transcription and translation of our CFPS reactions are controlled by stimuli-responsive riboswitches (RS). Via this scheme, adaptable material properties and functions are achieved in vitro via protein production from cell-free machinery administered through RS governance. Here, we present thermally or chemically-triggered riboswitches for orthogonal production of representative fluorescent protein products, as well functional proteins. To characterize the material properties of target proteins, we study the formation of polymerized actin shells to stabilize organically-encased droplets and span DIBs. We present a modified protocol for chemically-triggered actin polymerization as well as a thermally triggered actin RS. We characterize theophylline (TP)-triggered production of alpha hemolysin (α-HL) through CFPS and synthesized an organic-soluble trigger that can be sensed from the oil phase by a RS in an aqueous bioreactor droplet. We also demonstrate increased droplet conductivity when CFPS α-HL products are incorporated in DIBs. This interdisciplinary work involves cell culture, gene expression, organic synthesis, vesicle formation, protein quantification, tensiometry, droplet aspiration, microplate fluorescence/absorption experiments, fluorescent microscopy, and electrophysiology. This project is an essential design analysis for creating smart, soft materials using synthetic biology and provides motivation for artificial tissues capable of adapting in response to external stimuli.

Cell-free protein synthesis in microcompartments towards cell-cell communication.

In recent years, cell-free protein synthesis (CFPS) systems have been used to synthesize proteins, prototype genetic elements, manufacture chemicals, and diagnose diseases. These exciting, novel applications lead to a new wave of interest in the development of new CFPS systems that are derived from prokaryotic and eukaryotic organisms. The eukaryotic Pichia pastoris is emerging as a robust chassis host for recombinant protein production. To expand the current CFPS repertoire, we report here the development and optimization of a eukaryotic CFPS system, which is derived from a protease-deficient strain P. pastoris SMD1163. By developing a simple crude extract preparation protocol and optimizing CFPS reaction conditions, we were able to achieve superfolder green fluorescent protein (sfGFP) yields of 50.16 ± 7.49 μg/ml in 5 h batch reactions. Our newly developed P. pastoris CFPS system fits to the range of the productivity achieved by other eukaryotic CFPS platforms, normally ranging from several to tens of micrograms protein per milliliter in batch mode reactions. Looking forward, we believe that our P. pastoris CFPS system will not only expand the CFPS toolbox for synthetic biology applications, but also provide a novel platform for cost-effective, high-yielding production of complex proteins that need post-translational modification and functionalization.