Cell-free biology is the activation of biological processes without the use of intact living cells. It has been used for more than 50 years across the life sciences as a foundational research tool, but a recent technical renaissance has facilitated high-yielding (grams of protein per litre), cell-free gene expression systems from model bacteria, the development of cell-free platforms from non-model organisms and multiplexed strategies for rapidly assessing biological design. These advances provide exciting opportunities to profoundly transform synthetic biology by enabling new approaches to the model-driven design of synthetic gene networks, the fast and portable sensing of compounds, on-demand biomanufacturing, building cells from the bottom up, and next-generation educational kits. Cell-free gene expression systems have long been used to address fundamental research questions. Now, owing to technological advances, these systems are finding wider applications in the field of synthetic biology, including in biosensing, biomanufacturing, education and the design of gene networks.

Cell-free gene expression: an expanded repertoire of applications.

Cell-free protein synthesis (CFPS) is a platform technology that provides new opportunities for protein expression, metabolic engineering, therapeutic development, education, and more. The advantages of CFPS over in vivo protein expression include its open system, the elimination of reliance on living cells, and the ability to focus all system energy on production of the protein of interest. Over the last 60 years, the CFPS platform has grown and diversified greatly, and it continues to evolve today. Both new applications and new types of extracts based on a variety of organisms are current areas of development. However, new users interested in CFPS may find it challenging to implement a cell-free platform in their laboratory due to the technical and functional considerations involved in choosing and executing a platform that best suits their needs. Here we hope to reduce this barrier to implementing CFPS by clarifying the similarities and differences amongst cell-free platforms, highlighting the various applications that have been accomplished in each of them, and detailing the main methodological and instrumental requirement for their preparation. Additionally, this review will help to contextualize the landscape of work that has been done using CFPS and showcase the diversity of applications that it enables.

A User's Guide to Cell-Free Protein Synthesis.

Native cell-free transcription–translation systems offer a rapid route to characterize the regulatory elements (promoters, transcription factors) for gene expression from nonmodel microbial hosts, which can be difficult to assess through traditional in vivo approaches. One such host, Bacillus megaterium, is a giant Gram-positive bacterium with potential biotechnology applications, although many of its regulatory elements remain uncharacterized. Here, we have developed a rapid automated platform for measuring and modeling in vitro cell-free reactions and have applied this to B. megaterium to quantify a range of ribosome binding site variants and previously uncharacterized endogenous constitutive and inducible promoters. To provide quantitative models for cell-free systems, we have also applied a Bayesian approach to infer ordinary differential equation model parameters by simultaneously using time-course data from multiple experimental conditions. Using this modeling framework, we were able to infer previously unknown transcription factor binding affinities and quantify the sharing of cell-free transcription–translation resources (energy, ribosomes, RNA polymerases, nucleotides, and amino acids) using a promoter competition experiment. This allows insights into resource limiting-factors in batch cell-free synthesis mode. Our combined automated and modeling platform allows for the rapid acquisition and model-based analysis of cell-free transcription–translation data from uncharacterized microbial cell hosts, as well as resource competition within cell-free systems, which potentially can be applied to a range of cell-free synthetic biology and biotechnology applications.

https://www.pnas.org/content/pnas/115/19/E4340.full.pdf

Rapid acquisition and model-based analysis of cell-free transcription–translation reactions from nonmodel bacteria

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.

/pdf/cell-free-synthetic-biology-engineering-beyond-the-cell-27yyfyagk8.pdf

Cell-Free Synthetic Biology: Engineering Beyond the Cell

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 be thought of as programmable liquids, have removed many of these complexities and have brought about exciting opportunities for rational design and manipulation of biological systems. Here we review how these simple and accessible enzymatic systems are poised to accelerate the rate of advancement in synthetic biology and, more broadly, biotechnology.

/pdf/synthetic-biology-goes-cell-free-26yx37p7qe.pdf

Synthetic Biology Goes Cell-Free

Antibodies are well-established as therapeutics, and the preclinical and clinical pipeline of these important biologics is growing rapidly. Consequently, there is considerable interest in technologies to engineer and manufacture them. Mammalian cell culture is commonly used for production because eukaryotic expression systems have evolved complex and efficient chaperone systems for the folding of antibodies. However, given the ease and manipulability of bacteria, antibody discovery efforts often employ bacterial expression systems despite their limitations in generating high titers of functional antibody. Open-Cell Free Synthesis (OCFS) is a coupled transcription-translation system that has the advantages of prokaryotic systems while achieving high titers of antibody expression. Due to the open nature of OCFS, it is easily modified by chemical or protein additives to improve the folding of select proteins. As such, we undertook a protein additive screen to identify chaperone proteins that improve the folding and assembly of trastuzumab in OCFS. From the screen, we identified the disulfide isomerase DsbC and the prolyl isomerase FkpA as important positive effectors of IgG folding. These periplasmic chaperones function synergistically for the folding and assembly of IgG, and, when present in sufficient quantities, gram per liter IgG titers can be produced. This technological advancement allows the rapid development and manufacturing of immunoglobulin proteins and pushes OCFS to the forefront of production technologies for biologics.

Engineering toward a bacterial “endoplasmic reticulum” for the rapid expression of immunoglobulin proteins

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Engineering toward a bacterial “endoplasmic reticulum” for the rapid expression of immunoglobulin proteins