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BMC Biology Aug 2019Cell-free systems (CFS) have recently evolved into key platforms for synthetic biology applications. Many synthetic biology tools have traditionally relied on cell-based... (Review)
Review
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.
Topics: Biotechnology; Cell-Free System; Synthetic Biology
PubMed: 31395057
DOI: 10.1186/s12915-019-0685-x -
Molecular Plant Feb 2023
Topics: Cell-Free System; Proteins; Plants
PubMed: 36435966
DOI: 10.1016/j.molp.2022.11.013 -
Physical Biology May 2019
Topics: Cell-Free System; Synthetic Biology
PubMed: 31018188
DOI: 10.1088/1478-3975/ab1bed -
Current Opinion in Biotechnology Jun 2022Cell-free expression systems are becoming increasingly widely used due to their diverse applications in biotechnology. Despite this rapid expansion in adoption, many... (Review)
Review
Cell-free expression systems are becoming increasingly widely used due to their diverse applications in biotechnology. Despite this rapid expansion in adoption, many aspects of cell-free systems remain surprisingly poorly understood. Systems biology approaches make it possible to characterize cell-free systems deeply and broadly to better understand their underlying complexity. Here, we review recent systems biology studies that have provided insight into cell-free systems. We focus on characterization of the cell-free proteome, including its dependence on preparation protocol and host strain, as well as the cell-free metabolome and the relationship of endogenous metabolism to system performance. We conclude by highlighting promising future research directions.
Topics: Biotechnology; Cell-Free System; Metabolome; Proteome; Systems Biology
PubMed: 35247659
DOI: 10.1016/j.copbio.2022.102703 -
RNA (New York, N.Y.) Dec 2023Cell-free protein synthesis (CFPS) systems enable easy in vitro expression of proteins with many scientific, industrial, and therapeutic applications. Here we present an...
Cell-free protein synthesis (CFPS) systems enable easy in vitro expression of proteins with many scientific, industrial, and therapeutic applications. Here we present an optimized, highly efficient human cell-free translation system that bypasses many limitations of currently used in vitro systems. This CFPS system is based on extracts from human HEK293T cells engineered to endogenously express GADD34 and K3L proteins, which suppress phosphorylation of translation initiation factor eIF2α. Overexpression of GADD34 and K3L proteins in human cells before cell lysate preparation significantly simplifies lysate preparation. We find that expression of the GADD34 and K3L accessory proteins before cell lysis maintains low levels of phosphorylation of eIF2α in the extracts. During in vitro translation reactions, eIF2α phosphorylation increases moderately in a GCN2-dependent fashion that can be inhibited by GCN2 kinase inhibitors. This new CFPS system should be useful for exploring human translation mechanisms in more physiological conditions outside the cell.
Topics: Humans; HEK293 Cells; Phosphorylation; Proteins; Eukaryotic Initiation Factor-2; Protein Biosynthesis; Cell-Free System
PubMed: 37793791
DOI: 10.1261/rna.079825.123 -
The International Journal of... 2016Typically sperm nuclei are genetically inert and contain extremely compacted chromatin. Following fertilization, the first steps in their conversion to somatic nuclei... (Review)
Review
Typically sperm nuclei are genetically inert and contain extremely compacted chromatin. Following fertilization, the first steps in their conversion to somatic nuclei (male pronuclei) which will support further development involve chromatin decondensation and the formation of a new nuclear envelope. We have studied the reactivation of sea urchin sperm nuclei in a cell-free system derived from homogenates of activated sea urchin egg cytoplasm. The cell-free system has provided several novel insights including requirements for sperm-specific histone phosphorylation on N- and C-terminal extensions and disassembly of the sperm nuclear lamina for decondensation, the utilization of remnant regions of the sperm nuclear envelope to direct polarized binding and fusion of egg membranes to form the new nuclear envelope, and a role for phosphoinositide metabolism in initiation of membrane fusion through binding of a minor membrane fraction enriched in PtdIns (4,5)P, PLCγ and SFK1 which locally produces a fusigenic lipid, diacylglycerol.
Topics: Animals; Cell Nucleus; Cell-Free System; Chromatin; Male; Nuclear Envelope; Sea Urchins; Sperm-Ovum Interactions
PubMed: 27160073
DOI: 10.1387/ijdb.160077dp -
PloS One 2022Cell-free protein expression is increasingly becoming popular for biotechnology, biomedical and research applications. Among cell-free systems, the most popular one is...
Cell-free protein expression is increasingly becoming popular for biotechnology, biomedical and research applications. Among cell-free systems, the most popular one is based on Escherichia coli (E. coli). Endogenous nucleases in E. coli cell-free transcription-translation (TXTL) degrade the free ends of DNA, resulting in inefficient protein expression from linear DNA templates. RecBCD is a nuclease complex that plays a major role in nuclease activity in E. coli, with the RecB subunit possessing the actual nuclease activity. We created a RecB knockout of an E. coli strain optimized for cell-free expression. We named this new strain Akaby. We demonstrated that Akaby TXTL successfully reduced linear DNA degradations, rescuing the protein expression efficiency from the linear DNA templates. The practicality of Akaby for TXTL is an efficient, simple alternative for linear template expression in cell-free reactions. We also use this work as a model protocol for modifying the TXTL source E. coli strain, enabling the creation of TXTL systems with other custom modifications.
Topics: Cell-Free System; DNA; Escherichia coli; Escherichia coli Proteins; Exodeoxyribonuclease V
PubMed: 35390057
DOI: 10.1371/journal.pone.0266272 -
Cell Systems Jun 2021Molecular translation systems provide a genetically encoded framework for protein synthesis, which is essential for all life. Engineering these systems to incorporate... (Review)
Review
Molecular translation systems provide a genetically encoded framework for protein synthesis, which is essential for all life. Engineering these systems to incorporate non-canonical amino acids (ncAAs) into peptides and proteins has opened many exciting opportunities in chemical and synthetic biology. Here, we review recent advances that are transforming our ability to engineer molecular translation systems. In cell-based systems, new processes to synthesize recoded genomes, tether ribosomal subunits, and engineer orthogonality with high-throughput workflows have emerged. In cell-free systems, adoption of flexizyme technology and cell-free ribosome synthesis and evolution platforms are expanding the limits of chemistry at the ribosome's RNA-based active site. Looking forward, innovations will deepen understanding of molecular translation and provide a path to polymers with previously unimaginable structures and functions.
Topics: Amino Acids; Cell-Free System; Protein Biosynthesis; Proteins; Synthetic Biology
PubMed: 34139167
DOI: 10.1016/j.cels.2021.04.001 -
Current Opinion in Biotechnology Jun 2017The fields of biosensing and bioremediation leverage the phenomenal array of sensing and metabolic capabilities offered by natural microbes. Synthetic biology provides... (Review)
Review
The fields of biosensing and bioremediation leverage the phenomenal array of sensing and metabolic capabilities offered by natural microbes. Synthetic biology provides tools for transforming these fields through complex integration of natural and novel biological components to achieve sophisticated sensing, regulation, and metabolic function. However, the majority of synthetic biology efforts are conducted in living cells, and concerns over releasing genetically modified organisms constitute a key barrier to environmental applications. Cell-free protein expression systems offer a path towards leveraging synthetic biology, while preventing the spread of engineered organisms in nature. Recent efforts in the areas of cell-free approaches for sensing, regulation, and metabolic pathway implementation, as well as for preserving and deploying cell-free expression components, embody key steps towards realizing the potential of cell-free systems for environmental sensing and remediation.
Topics: Animals; Biodegradation, Environmental; Biosensing Techniques; Cell-Free System; Metabolic Networks and Pathways; Protein Biosynthesis; Synthetic Biology; Transcription, Genetic
PubMed: 28226291
DOI: 10.1016/j.copbio.2017.01.010 -
Briefings in Bioinformatics Mar 2019Disease diagnosis using cell-free DNA (cfDNA) has been an active research field recently. Most existing approaches perform diagnosis based on the detection of sequence... (Review)
Review
Disease diagnosis using cell-free DNA (cfDNA) has been an active research field recently. Most existing approaches perform diagnosis based on the detection of sequence variants on cfDNA; thus, their applications are limited to diseases associated with high mutation rate such as cancer. Recent developments start to exploit the epigenetic information on cfDNA, which could have substantially wider applications. In this work, we provide thorough reviews and discussions on the statistical method developments and data analysis strategies for using cfDNA epigenetic profiles, in particular DNA methylation, to construct disease diagnostic models. We focus on two important aspects: marker selection and prediction model construction, under different scenarios. We perform simulations and real data analysis to compare different approaches, and provide recommendations for data analysis.
Topics: Cell-Free System; DNA Methylation; Epigenomics; Humans
PubMed: 29672679
DOI: 10.1093/bib/bby029