Search Results (43)

Synthetic biology is reshaping in vitro diagnostics (IVD) by enabling programmable and modular biosensing elements that can be integrated into point-of-care testing (POCT) platforms. Compared with conventional assays that depend on fixed chemistries and centralized instrumentation, synthetic biology-based systems offer adaptable molecular recognition, tunable signal processing, and flexible readout formats for decentralized diagnostics. In this review, we present synthetic biology-enabled IVD as programmable biosensing platforms organized into four functional layers: molecular recognition, signal transduction and amplification, output generation, and system integration. We discuss four major enabling modules, including cell-free protein synthesis (CFPS) systems, aptamer and riboswitch sensors, CRISPR-Cas diagnostic platforms, and microfluidic integration technologies. We summarize representative clinical applications from 2021 to 2025 in infectious disease detection, cancer biomarker analysis, and drug metabolism/toxicity screening. In addition, we examine practical considerations beyond analytical sensitivity, including matrix tolerance, workflow complexity, manufacturability, quantitative capability, and regulatory readiness. Finally, we highlight future directions for programmable diagnostics, including AI-assisted biosensor design, multimodal readouts, interoperable platform architectures, and real-world clinical validation. Full article

Heme proteins are central to metabolism and stress responses but remain challenging to express recombinantly due to cytotoxicity and folding constraints. Phytoglobins (Pgbs) exemplify these difficulties, as expression protocols often fail to translate across protein species. Here, we used a cell-free protein synthesis (CFPS) platform powered by digital microfluidics to screen expression determinants for sugar beet Pgb 1.2 (BvPgb 1.2), its C86A variant, and three of eight newly identified oat Pgbs (AsPgbs), including their cysteine-to-alanine substituted variants. Benchmarking with multiple solubility tags and cell-free blends revealed protein- and variant-specific preferences, with alanine substitutions frequently improving expression and purification yields. Oxidative additives such as glutathione disulfide, alone or combined with protein disulfide isomerase, consistently enhanced production, underscoring the importance of redox environments for Pgb stability. Two selected variants were scaled up and yielded putative soluble apo-form proteins. The results highlight how CFPS enables rapid, parallelized identification of expression requirements while uncovering the role of conserved cysteines and redox conditions in Pgb biogenesis. Full article

Cell-free protein synthesis (CFPS) has transformed protein production capabilities by eliminating cellular constraints, enabling the rapid expression of difficult-to-produce proteins in an open, customizable environment. As CFPS applications expand from fundamental research to industrial production, therapeutic manufacturing, and point-of-care diagnostics, the diverse array of reactor formats has become increasingly important yet challenging to navigate. This review examines the evolution and characteristics of thirteen major CFPS reactor formats, from traditional batch systems to advanced platforms. The historical development of CFPS reactors from the 1960s to present day is presented. Additionally, for each format, operational principles, advantages, limitations, and notable applications are evaluated. The review concludes with a comparative assessment of reactor performance across critical parameters, including productivity, scalability, technical complexity, environmental stability, and application suitability. To our knowledge this structured analysis is the first to focus predominantly on the various reactor formats of cell-free systems and to provide a guide to assist researchers in choosing the reactor type that best fits their specific applications. Full article

Glutamine is an essential biomolecule that plays a pivotal role in many diseases, such as cancer, where it can serve as fuel for rapid proliferation. Treatments for these diseases can be monitored and optimized through the detection of glutamine, though standard glutamine detection procedures are costly and require complex instrumentation. Cell-free protein synthesis (CFPS) has recently enabled a paper-based, colorimetric glutamine sensor that carries the potential to increase test accessibility while dramatically reducing consumer cost to enable at-home, rapid treatment monitoring. Test sensitivity remained limited by residual assay background, thus motivating this work where CFPS reactions traditionally formulated with glutamate salts were compared to systems using alternative salts, including aspartate, acetate, citrate, and sulfate, to reduce the background generation of glutamine. This led to the discovery of a novel aspartate-based CFPS system that boasts a high signal strength and indetectable background noise over 225 min. Acetate-, citrate-, and sulfate-based systems also yielded zero background glutamine detection but at a lower signal response compared to the aspartate-based system. These findings mark crucial advancements in producing a cost-effective, simple glutamine monitor while simultaneously showcasing the adaptability of CFPS’s open reaction environment for solving complex challenges in next-generation biosensor development. Full article

Centralised cell-based biomanufacturing severely limits applicability in low-resource and extreme environments, where a largely untreated human population is present. Cell-free protein synthesis (CFPS) can surpass many of these limitations, due to its flexibility and low maintenance. After initial optimisation for high-level expression, we conceptualised CFPS platforms composed of lyophilised pellets and cellulose stacks for ease of storage and distribution. The latter platform consisted of lyophilised components on cellulose discs, which were layered and rehydrated to kickstart protein synthesis. Such paper-encompassed reactions were capable of robust expression, where the system can be modulated by simply changing the DNA layer. Using an initial screening design followed by a minimalistic design of experiments approach, we were able to improve the shelf life of lyophilised CFPS at room temperature from <1 week to 100% preservation at month 1. We anticipate that our strategy will enable quicker and more efficient stability optimisation for sustainable applications in all environments. Full article

Enzymes play an essential role in many different industries; however, their operating conditions are limited due to the loss of enzyme activity in the presence of proteases and at temperatures significantly above physiological conditions. One way to improve the stability of these enzymes against high temperatures and proteases is to encapsulate them in protective shells or virus-like particles. This work presents a streamlined, three-step, cell-free protein synthesis (CFPS) procedure that enables rapid in vitro enzyme production, targeted encapsulation in protective virus-like particles (VLPs), and facile purification using a 6× His-tag fused to the VLP coat protein. This process is performed in under 12 h and overcomes several limitations of enzyme encapsulation, such as the control of packing density, speed, and complexity of the process. Here, we encapsulate the enzyme Candida antarctica lipase B in the VLP from the bacteriophage Qβ, while in the presence of a linking RNA aptamer. The encapsulated enzymes largely retained their activity in comparison to the free enzymes. Additionally, when subjected to 90 °C temperatures or 5 h incubation with proteases, the encapsulated enzymes maintained their activity, whereas the free enzymes lost their activity. In this work, we also demonstrate control over packing density by achieving packing densities of 4.7 and 6.5 enzymes per VLP based off the concentration of enzyme added to the encapsulation step. Full article

Background/Objectives: Cancer is a leading cause of death. However, recently developed immunotherapies have shown significant promise to improve cancer treatment outcomes and survival rates. Pembrolizumab, a cancer immunotherapy drug, enables a strong T-cell response specifically targeting cancer cells to improve patient outcomes in more than 16 types of cancer. The increasing demand for pembrolizumab, the highest selling drug in 2023, increases global dependence on drug production, which can be vulnerable to supply chain disruptions. Methods: Cell-free protein synthesis (CFPS) is a rapid in vitro protein production method that could provide the production of an immunotherapy drug in an emergency and could facilitate on-demand production of the therapeutic at the point of care if needed. Furthermore, CFPS has potential as a production platform of biosimilars, as the patent for pembrolizumab is set to expire in 2028. Results: This work presents the design, synthesis, and target-binding affinity of a novel single-chain variable fragment of pembrolizumab (Pem-scFv) using CFPS. The CFPS production of Pem-scFv also enables the direct optimization of synthesis reaction composition and expression conditions. The conditions of 30 °C, 35% (v/v) cell extract, and an oxidizing redox environment resulted in the highest Pem-scFv soluble yield of 442 µg/mL. An affinity assay demonstrated significant binding between the CFPS-produced Pem-scFv and the PD-1 target. Computational simulations of Pem-scFv folding and binding corroborate the experimental results. Full article

Naturally occurring protein toxins can derive from bacteria, fungi, plants, and animal venom. Traditionally, toxins are known for their destructive effects on host cells. Despite, and sometimes even because of, these harmful effects, toxins have been used for medical benefits. The prerequisite for the development of toxin-based medications or treatments against toxins is thorough knowledge about the toxin and its underlying mechanism of action. Thus, the toxin of interest must be synthesized. Traditional cell-based production requires high laboratory safety standards and often results in a low total protein yield due to the toxin’s harmful, cytotoxic nature. These drawbacks can be circumvented by using cell-free protein synthesis (CFPS), a highly adaptable platform technology relying on cell lysates rather than living cells. This review discusses the current advances in cell-free synthesis of protein toxins as well as their uses and applications for pharmaceutical and diagnostic purposes. Full article

Background/Objectives: Antimicrobial-resistant (AMR) pathogens represent a serious threat to public health, particularly in food production systems where antibiotic use remains widespread. As a result, alternative antimicrobial treatments to antibiotics are essential for effectively managing bacterial infections. This study aimed to identify and characterize novel antimicrobial peptides produced by bacteria, known as bacteriocins, as well as to recognize safe bacteriocin-producing strains, sourced from poultry slaughterhouse effluents. Methods: A total of 864 bacterial isolates were collected across eight stages of a poultry slaughter line and screened for antimicrobial activity against Gram-positive and Gram-negative indicator strains. Whole-genome sequencing (WGS) was performed on 12 selected strains, including Enterococcus faecium (6 isolates), Lactococcus lactis (1 isolate), Lactococcus garvieae (1 isolate) and Escherichia coli (4 isolates). The presence of bacteriocin gene clusters (BGC), antibiotic resistance genes (ARG), and virulence factors (VF) was analyzed. The antimicrobial activity of a novel bacteriocin was further evaluated using in vitro cell-free protein synthesis (IV-CFPS). Results: WGS revealed multiple BGCs, including a novel class IId bacteriocin, lactococcin P1A (LcnP1A), in L. lactis SWD9. LcnP1A showed antimicrobial activity against various indicator strains, including Listeria monocytogenes. While most bacteriocin-encoding strains harbored ARGs and VFs, E. faecium SWG6 was notable for its absence of ARGs and minimal VFs, highlighting its potential as a probiotic. Conclusions: These findings underscore the importance of discovering novel bacteriocins and safer bacteriocin producing strains to address antimicrobial resistance in the food chain. Further research would validate the efficacy of both the novel lactococcin P1A bacteriocin and the E. faecium SWG6 isolate for application in processed food and animal production systems. Full article

Antimicrobial resistance (AMR) poses a significant challenge to animal production due to the widespread use of antibiotics. Therefore, there is an urgent need for alternative antimicrobial strategies to effectively manage bacterial infections, protect animal health, and reduce reliance on antibiotics. This study evaluated the use of emerging approaches and procedures for the isolation, identification, and characterization of bacteriocin-producing bacteria and their bacteriocins, sourced from the gastrointestinal tract (GIT) of meat-producing pigs. Out of 2056 isolates screened against Gram-positive and Gram-negative indicator strains, 20 of the most active antimicrobial isolates were subjected to whole genome sequencing (WGS) for the prediction of coding DNA sequences (CDS) and the identification of bacteriocin gene clusters (BGC) and their functions. The use of an in vitro cell-free protein synthesis (IV-CFPS) protocol and the design of an IV-CFPS coupled to a split-intein mediated ligation (IV-CFPS/SIML) procedure made possible the evaluation of the production and antimicrobial activity of described and putatively novel bacteriocins. A colony MALDI-TOF MS procedure assisted in the identification of class I, II, and III lanthipeptides. MALDI-TOF MS and a targeted proteomics, combined with a massive peptide analysis (LC-MS/MS) approach, has proven valuable for the identification and biochemical characterization of previously described and novel bacteriocins encoded by the isolated bacteriocin-producing strains. Full article

Background: Vaccine development against Chlamydia, a prevalent sexually transmitted infection (STI), is imperative due to its global public health impact. However, significant challenges arise in the production of effective subunit vaccines based on recombinant protein antigens, particularly with membrane proteins like the Major Outer Membrane Protein (MOMP). Methods: Cell-free protein synthesis (CFPS) technology is an attractive approach to address these challenges as a method of high-throughput membrane protein and protein complex production coupled with nanolipoprotein particles (NLPs). NLPs provide a supporting scaffold while allowing easy adjuvant addition during formulation. Over the last decade, we have been working toward the production and characterization of MOMP-NLP complexes for vaccine testing. Results: The work presented here highlights the expression and biophysical analyses, including transmission electron microscopy (TEM) and dynamic light scattering (DLS), which confirm the formation and functionality of MOMP-NLP complexes for use in animal studies. Moreover, immunization studies in preclinical models compare the past and present protective efficacy of MOMP-NLP formulations, particularly when co-adjuvanted with CpG and FSL1. Conclusion: Ex vivo assessments further highlight the immunomodulatory effects of MOMP-NLP vaccinations, emphasizing their potential to elicit robust immune responses. However, further research is warranted to optimize vaccine formulations further, validate efficacy against Chlamydia trachomatis, and better understand the underlying mechanisms of immune response. Full article

Background:Chlamydia trachomatis is the most prevalent bacterial sexually transmitted pathogen in humans worldwide. Since chlamydial infection is largely asymptomatic with the potential for serious complications, a preventative vaccine is likely the most viable long-term answer to this public health threat. Cell-free protein synthesis (CFPS) utilizes the cellular protein manufacturing machinery decoupled from the requirement for maintaining cellular viability, offering the potential for flexible, rapid, and decentralized production of recombinant protein vaccine antigens. Methods: Here, we use CFPS to produce the full-length putative chlamydial type three secretion system (T3SS) needle-tip protein, CT584, for evaluation as a vaccine antigen in mouse models. High-speed atomic force microscopy (HS-AFM) (RIBM, Tsukuba, Japan) imaging and computer simulations confirm that CFPS-produced CT584 retains a native-like structure prior to immunization. Female mice were primed with CT584 adjuvanted with CpG-1826 intranasally (i.n.) or CpG-1826 + Montanide ISA 720 intramuscularly (i.m.), followed four weeks later by an i.m. boost before respiratory challenge with 10⁴ inclusion forming units (IFU) of Chlamydia muridarum. Results: Immunization with CT584 generated robust antibody responses but weak cell-mediated immunity and failed to protect against i.n. challenge as demonstrated by body weight loss, increased lung weights, and the presence of high numbers of IFUs in the lungs. Conclusion: While CT584 was not a protective vaccine candidate, the speed and flexibility with which CFPS can be used to produce other potential chlamydial antigens make it an attractive technique for antigen production. Full article

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. Full article

Specialized cancer treatments have the potential to exploit glutamine dependence to increase patient survival rates. Glutamine diagnostics capable of tracking a patient’s response to treatment would enable a personalized treatment dosage to optimize the tradeoff between treatment success and dangerous side effects. Current clinical glutamine testing requires sophisticated and expensive lab-based tests, which are not broadly available on a frequent, individualized basis. To address the need for a low-cost, portable glutamine diagnostic, this work engineers a cell-free glutamine biosensor to overcome assay background and signal-to-noise limitations evident in previously reported studies. The findings from this work culminate in the development of a shelf-stable, paper-based, colorimetric glutamine test with a high signal strength and a high signal-to-background ratio for dramatically improved signal resolution. While the engineered glutamine test is important progress towards improving the management of cancer and other health conditions, this work also expands the assay development field of the promising cell-free biosensing platform, which can facilitate the low-cost detection of a broad variety of target molecules with high clinical value. Full article

The rise of antimicrobial resistance poses a significant global health threat, necessitating urgent efforts to identify novel antimicrobial agents. In this study, we undertook a thorough screening of soil-derived bacterial isolates to identify candidates showing antimicrobial activity against Gram-positive bacteria. A highly active antagonistic isolate was initially identified as Bacillus altitudinis ECC22, being further subjected to whole genome sequencing. A bioinformatic analysis of the B. altitudinis ECC22 genome revealed the presence of two gene clusters responsible for synthesizing two circular bacteriocins: pumilarin and a novel circular bacteriocin named altitudin A, alongside a closticin 574-like bacteriocin (CLB) structural gene. The synthesis and antimicrobial activity of the bacteriocins, pumilarin and altitudin A, were evaluated and validated using an in vitro cell-free protein synthesis (IV-CFPS) protocol coupled to a split-intein-mediated ligation procedure, as well as through their in vivo production by recombinant E. coli cells. However, the IV-CFPS of CLB showed no antimicrobial activity against the bacterial indicators tested. The purification of the bacteriocins produced by B. altitudinis ECC22, and their evaluation by MALDI-TOF MS analysis and LC-MS/MS-derived targeted proteomics identification combined with massive peptide analysis, confirmed the production and circular conformation of pumilarin and altitudin A. Both bacteriocins exhibited a spectrum of activity primarily directed against other Bacillus spp. strains. Structural three-dimensional predictions revealed that pumilarin and altitudin A may adopt a circular conformation with five- and four-α-helices, respectively. Full article