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![Overview of the use of biosensors in CFS. The general workflow usually involves in silico design of gene circuits encoding biosensors and reporter proteins, followed by chemical synthesis of such circuits. Meanwhile, patient or environmental samples are collected, target analytes are extracted, and, in some cases, amplified. The gene circuits and target analytes are then added to CFS. Examples of biosensors in CFS have included a) mercury (II) detection using the MerR repressor[45], b) viral and bacterial nucleic acid sensing using toehold switch-based sensors [46, 50, 59], c) identification of P. aeruginosa infection by its quorum sensing molecule, 3-oxo-C12-HSL, using the LasRV sensor [61] and d) recognition of an endocrine-disrupting compound by utilizing an allosterically activated fusion protein containing the ligand binding domain of a human estrogen receptor [62, 63]. Reporters (e.g., colorimetric or fluorescent) can then produced, contingent upon analyte detection, enabling clinical diagnosis (e.g., using standard spectrophotometers)](publication/335075642/figure/fig4/AS:962841397960711@1606570686482/Overview-of-the-use-of-biosensors-in-CFS-The-general-workflow-usually-involves-in-silico.png)
Overview of the use of biosensors in CFS. The general workflow usually involves in silico design of gene circuits encoding biosensors and reporter proteins, followed by chemical synthesis of such circuits. Meanwhile, patient or environmental samples are collected, target analytes are extracted, and, in some cases, amplified. The gene circuits and target analytes are then added to CFS. Examples of biosensors in CFS have included a) mercury (II) detection using the MerR repressor[45], b) viral and bacterial nucleic acid sensing using toehold switch-based sensors [46, 50, 59], c) identification of P. aeruginosa infection by its quorum sensing molecule, 3-oxo-C12-HSL, using the LasRV sensor [61] and d) recognition of an endocrine-disrupting compound by utilizing an allosterically activated fusion protein containing the ligand binding domain of a human estrogen receptor [62, 63]. Reporters (e.g., colorimetric or fluorescent) can then produced, contingent upon analyte detection, enabling clinical diagnosis (e.g., using standard spectrophotometers)
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Cell-free systems (CFS) have recently evolved into key platforms for synthetic biology applications. Many synthetic biology tools have traditionally relied on cell-based systems, and while their adoption has shown great progress, the constraints inherent to the use of cellular hosts have limited their reach and scope. Cell-free systems, which can b...
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Cell-free protein expression systems have been widely used for synthetic biology and metabolic engineering applications in recent years. Yet little is known about protein expression in the cell-free systems. Here we take a systems approach to uncover underlying dynamics of cell-free protein expression. We construct a set of T7 promoter variants to...
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... Cell-free protein synthesis (CFPS) systems provide a promising alternative for the rapid expression of target proteins (Gregorio et al., 2019;Perez et al., 2016;Zemella et al., 2015). Utilizing an appropriate DNA template facilitates the synthesis of target proteins within a few hours, thereby enabling the study of challenging proteins, including those with disulfide bonds that are sometimes difficult to express in conventional cellbased systems (Chiba et al., 2021;Stech et al., 2017;Tinafar et al., 2019). CFPS has been successfully used to produce various antibody fragments and full-length IgGs (Buntru et al., 2015;Martin et al., 2017;Murakami et al., 2019;Xu et al., 2015). ...
Antibodies are critical tools in medicine and research, and their affinity for their target antigens is a key determinant of their efficacy. Traditional antibody affinity maturation and interaction analyses are often hampered by time‐consuming steps such as cloning, expression, purification, and interaction assays. To address this, we have developed FASTIA (Fast Affinity Screening Technology for Interaction Analysis), a novel platform that integrates rapid gene fragment preparation, cell‐free protein synthesis, and bio‐layer interferometry with non‐regenerative analysis. Using this approach, we can analyze the intermolecular interactions of over 20 variants over 2 days, requiring only the parent protein expression plasmid and basic equipment. We have demonstrated the ability of FASTIA to discriminate between single‐domain antibody variants with different binding affinities using the anti‐HEL VHH antibody D2‐L29, and mapped the results to the crystal structure to identify key interaction sites. FASTIA provides results comparable to those obtained using traditional methods. Our system bypasses the need for genetic engineering facilities and can be easily adopted by laboratories, accelerating the protein engineering and optimization processes. In addition, FASTIA is applicable to other protein–protein interactions, making it a versatile tool for studying molecular recognition. FASTIA facilitates efficient affinity maturation, protein engineering, and analysis of protein–protein interactions. This provides a rapid and accessible route for improving antibodies and a broader understanding of protein interactions.
... 11−13 CFPS also finds utility in enzyme variant evolution and screening, 14 medical diagnostics and biosensor design, 15 and artificial cell construction, 16 among other applications. 9,17,18 Recently, CFPS has been integrated with diverse strategies, such as chemistry and materials, to enhance its performance in biocatalysis and biotransformation. Through CFPS, triblock copolymer-based aqueous−organic biphasic systems, 19 light/heating-controlled cell-free reactions, 5 and enzyme-polymer-conjugate-based Pickering emulsions 20 have been developed for efficient cell-free expression and biotransformation. ...
Cell-free protein synthesis (CFPS) has proven invaluable for expressing a wide array of proteins and enzymes, boasting significant advantages, such as facile manipulation, rapid mass transfer, and high productivity. However, traditional cell-free expression systems typically operate in batch format reactions, posing challenges for enzyme reuse in biocatalysis and rendering supplemented gene templates nonrecyclable. To overcome this limitation, we introduce a chitin-functionalized CFPS system designed to immobilize in vitro-expressed enzymes and gene templates for sustainable biocatalysis and protein synthesis. At its core, this system capitalizes on the strong binding affinity between a chitin-binding domain (ChBD) and crystalline chitin nanofibers (ChNFs). Specifically, we engineer ChBD fusion with target proteins and enzymes, allowing for their cell-free expression and in situ immobilization on ChNFs. This facilitates the effortless recycling of enzymes for multiple biocatalytic reactions, while ChNFs-immobilized enzymes can also be deployed in continuous flow biocatalysis setups. Leveraging the ChBD-ChNF pairing, gene templates can likewise be immobilized and recycled for sustained gene expression. Our results demonstrate that the chitin-functionalized cell-free system significantly enhances CFPS performance through immobilization with ChNF materials. This work underscores the flexibility and robustness of cell-free systems, which can seamlessly integrate with advanced techniques from fields such as chemistry and materials science for impactful applications.
... For example, Engineering at cellular level is problematic particularly because of membrane barriers (hinder optimization of synthetic process, cause incompatibility, variability issues) and complicated metabolic pathways ( Jiang et al., 2018;Yue et al., 2019). Moreover, either Suitable vectors are required for carrying genetic instructions to cells that should be maintained through chromosomal integration or selectable marker expression to allow instructions to be evaluated (Tinafar et al., 2019). Other limitations of cell-based systems are limited product yield due to toxicity effects, synthesis of competing by-products, difficulty in optimization of labscale cultures to commercial production-scale because of variable fermentation conditions and constraints in downstream processing in case of intra-cellular accumulation of product (Takors, 2012). ...
... In CFPS, the DNA encoding the desired protein is incubated with a cell extract, appropriately supplemented with amino acids, tRNA etc.-the majority of examples discussed here use E. coli extracts (Gregorio et al., 2019;Perez et al., 2016). CFPS using extracts from other prokaryotic and eukaryotic organisms is also possible, with a variety of associated advantages and disadvantages (Batista et al., 2021;Moore et al., 2021;Tinafar et al., 2019). ...
In protein design, the ultimate test of success is that the designs function as desired. Here, we discuss the utility of cell free protein synthesis (CFPS) as a rapid, convenient and versatile method to screen for activity. We champion the use of CFPS in screening potential designs. Compared to in vivo protein screening, a wider range of different activities can be evaluated using CFPS, and the scale on which it can easily be used—screening tens to hundreds of designed proteins—is ideally suited to current needs. Protein design using physics‐based strategies tended to have a relatively low success rate, compared with current machine‐learning based methods. Screening steps (such as yeast display) were often used to identify proteins that displayed the desired activity from many designs that were highly ranked computationally. We also describe how CFPS is well‐suited to identify the reasons designs fail, which may include problems with transcription, translation, and solubility, in addition to not achieving the desired structure and function.
... The fact that animal cells-including those of fish and aquatic invertebrates-are incapable of synthesizing omega-3 fats de novo means that producers of cell-cultivated seafood will need to acquire appropriate sources of omega-3 fatty acids as ingredients. These sources could include farming of microalgae [96], precision fermentation [97,98], plant molecular farming [99,100], or cellfree systems [101]. However, this latter strategy has not yet been explored for omega-3 production to our knowledge, and the former three strategies will still require substantial effort before they can be scaled to the levels that may be required to support the cell-cultivated seafood industry. ...
... In addition, cellular engineering may provide a potential solution to enhance the accumulation and stability of omega-3 fats. These approaches may include the use of exogenous reactive oxygen scavengers in the media to promote cell proliferation and suppress oxidation processes [101], as well as genetic modifications to over-express antioxidant genes, such as superoxide dismutase (SOD). Furthermore, cellular engineering approaches also enable the design of media compositions to promote the synthesis of omega-3 fats [103]. ...
The demand for fish protein continues to increase and currently accounts for 17% of total animal protein consumption by humans. About 90% of marine fish stocks are fished at or above maximum sustainable levels, with aquaculture propagating as one of the fastest growing food sectors to address some of this demand. Cell-cultivated seafood production is an alternative approach to produce nutritionally-complete seafood products to meet the growing demand. This cellular aquaculture approach offers a sustainable, climate resilient and ethical biotechnological approach as an alternative to conventional fishing and fish farming. Additional benefits include reduced antibiotic use and the absence of mercury. Cell-cultivated seafood also provides options for the fortification of fish meat with healthier compositions, such as omega-3 fatty acids and other beneficial nutrients through scaffold, media or cell approaches. This review addresses the biomaterials, production processes, tissue engineering approaches, processing, quality, safety, regulatory, and social aspects of cell-cultivated seafood, encompassing where we are today, as well as the road ahead. The goal is to provide a roadmap for the science and technology required to bring cellular aquaculture forward as a mainstream food source.
... They are especially promising because protein synthesis is often facilitated in only a few hours. A range of cell-free systems are commercially available and optimized to carry out protein production with simple protocols, allowing parallelization, and thus enabling a high throughput [17][18][19][20]. Lastly, the generation of genetically modified organisms is circumvented. ...
Venoms are a complex cocktail of potent biomolecules and are present in many animal lineages. Owed to their translational potential in biomedicine, agriculture and industrial applications, they have been targeted by several biodiscovery programs in the past. That said, many venomous animals are relatively small and deliver minuscule venom yields. Thus, the most commonly employed activity-guided biodiscovery pipeline cannot be applied effectively. Cell-free protein production may represent an attractive tool to produce selected venom components at high speed and without the creation of genetically modified organisms, promising rapid and highly efficient access to biomolecules for bioactivity studies. However, these methods have only sporadically been used in venom research and their potential remains to be established. Here, we explore the ability of a prokaryote-based cell-free system to produce a range of venom toxins of different types and from various source organisms. We show that only a very limited number of toxins could be expressed in small amounts. Paired with known problems to facilitate correct folding, our preliminary investigation underpins that venom-tailored cell-free systems probably need to be developed before this technology can be employed effectively in venom biodiscovery.
... To characterize the performance of the engineered BenM P196D biosensor in other contexts and to enable future engineering, we implemented a cell-free reaction system to express the BenM biosensors. Cell-free systems provide a variety of advantages to the whole-cell sensors such as overcoming constraints like ligand uptake limitations and differences between cellular contexts [32,33]. We implemented a cell-free system utilizing a previously developed high-copy vector expressing WT BenM, or BenM P196D , named here as pJKR_WT and pJKR_P196D, respectively [34,35]. ...
Transcription factor (TF)-based biosensors that connect small-molecule sensing with readouts such as fluorescence have proven to be useful synthetic biology tools for applications in biotechnology. However, the development of specific TF-based biosensors is hindered by the limited repertoire of TFs specific for molecules of interest since current construction methods rely on a limited set of characterized TFs. In this study, we present an approach for engineering the specificity of TFs through a computation-based workflow using molecular docking that enables targeted alteration of TF ligand specificity. Using this method, we engineer the LysR family BenM TF to alter its specificity from its cognate ligand cis,cis-muconic acid to adipic acid through a single amino acid substitution identified by our computational workflow. When implemented in a cell-free system, the engineered biosensor shows higher ligand sensitivity, expanding the potential applications of this circuit. We further investigate ligand binding through molecular dynamics to analyze the substitution, elucidating the impact of modulating a single amino acid position on the mechanism of BenM ligand binding. This study represents the first application of biomolecular modeling methods for altering BenM specificity and for gaining insights into how mutations influence the structural dynamics of BenM. Such methods can potentially be applied to other TFs to alter specificity and analyze the dynamics responsible for these changes, highlighting the applicability of computational tools for informing experiments. In addition, our developed adipic acid biosensor can be applied for the identification and engineering of enzymes to produce adipic acid.
... 13,14 Since cell-free reactions do not contain living cells, unlike whole-cell systems, they do not need to be cultured, do not need maintenance by specialized equipment, and do not require biocontainment. 15 Here, we demonstrate the use of the CFPS platform BioBits aboard the International Space Station (ISS), enabling the development of technologies that may resolve long-standing challenges in space and on Earth. BioBits is an ideal synthetic biology tool for low-resource environments as it is not only low-cost and portable but also freezedried for long-term stability ( Figure 1A). ...
Cell-free protein synthesis (CFPS) is a rapidly maturing in vitro gene expression platform that can be used to transcribe and translate nucleic acids at the point of need, enabling on-demand synthesis of peptide-based vaccines and biotherapeutics as well as the development of diagnostic tests for environmental contaminants and infectious agents. Unlike traditional cell-based systems, CFPS platforms do not require the maintenance of living cells and can be deployed with minimal equipment; therefore, they hold promise for applications in low-resource contexts, including spaceflight. Here, we evaluate the performance of the cell-free platform BioBits aboard the International Space Station by expressing RNA-based aptamers and fluorescent proteins that can serve as biological indicators. We validate two classes of biological sensors that detect either the small-molecule DFHBI or a specific RNA sequence. Upon detection of their respective analytes, both biological sensors produce fluorescent readouts that are visually confirmed using a hand-held fluorescence viewer and imaged for quantitative analysis. Our findings provide insights into the kinetics of cell-free transcription and translation in a microgravity environment and reveal that both biosensors perform robustly in space. Our findings lay the groundwork for portable, low-cost applications ranging from point-of-care health monitoring to on-demand detection of environmental hazards in low-resource communities both on Earth and beyond.
... Synthetic cells hold the potential to advance precision medicine, increase access to pharmaceuticals in underserved communities, and transform therapeutic production processes globally (7,8). However, because artificial cells are an emerging technology, they still await lengthy scale up of the manufacturing and testing protocols. ...
... Synthetic cells offer opportunities to treat rare diseases, improve treatment efficacy and engineer life-saving therapeutics, much like vaccines (7,8,16). At the moment, however, they lack regulatory guidelines (9). ...
Synthetic cells are a novel class of cell-like bioreactors, offering the potential for unique advancements in synthetic biology and biomedicine. To realize the potential of those technologies, synthetic cell-based drugs need to go through the drug approval pipeline. Here, we discussed several regulatory challenges, both unique to synthetic cells, as well as challenges typical for any new biomedical technology. Overcoming those difficulties could bring transformative therapies to the market and will create a path to the development and approval of cutting-edge synthetic biology therapies.
Graphical Abstract
... The affordable and widely applicable biotechnologies that can be developed using CFPS platforms hold immense potential in low-resource settings, including in space (13,14). Since cell-free reactions do not contain living cells, unlike whole-cell systems, they do not need to be cultured, do not need maintenance by specialized equipment, and do not require biocontainment (15). Here, we demonstrate use of the CFPS platform BioBits® aboard the International Space Station (ISS), enabling the development of technologies that may resolve long-standing challenges in space and on Earth. ...
Cell-free protein synthesis (CFPS) is a rapidly maturing in vitro gene expression platform that can be used to transcribe and translate nucleic acids at the point of need, enabling on-demand synthesis of peptide-based vaccines and biotherapeutics, as well as the development of diagnostic tests for environmental contaminants and infectious agents. Unlike traditional cell-based systems, CFPS platforms do not require the maintenance of living cells and can be deployed with minimal equipment; therefore, they hold promise for applications in low-resource contexts, including spaceflight. Here we evaluate the performance of cell-free BioBits® platform aboard the International Space Station by expressing RNA-based aptamers and fluorescent proteins that can serve as biological indicators. We validate two classes of biological sensors that detect either the small molecule DFHBI or a specific RNA sequence. Upon detection of their respective analytes, both biological sensors produce fluorescent readouts that are visually confirmed using a handheld fluorescence viewer and imaged for quantitative analysis. Our findings provide insight into the kinetics of cell-free transcription and translation in a microgravity environment and reveal that both biosensors perform robustly in space. Our findings lay the groundwork for portable, low-cost applications ranging from point-of-care health monitoring to on-demand detection of environmental hazards in low-resource communities both on Earth and beyond.
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