Cell-free systems in the new age of synthetic biology

Villarreal, Fernando; Tan, Cheemeng

doi:10.1007/s11705-017-1610-x

Cited by 23 publications

(10 citation statements)

References 80 publications

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“…The dynamics and behaviors of gene circuits, including multistage cascades, gates, oscillators and negative feedback loops have been investigated using an E. coli -based protein synthesis system ( 10 , 14 ). Moreover, vesicles containing E. coli -based cell-free reactions have been established as an artificial cell platform ( 15 , 16 ). In contrast to E. coli -based systems, mammalian CFE systems present greater complexity and are less well-understood; however, mammalian CFE systems have increasingly gained researchers’ interests due to their ability to carry out post- and co-translational modifications ( 17 , 18 ).…”

Section: Introductionmentioning

confidence: 99%

Simultaneous monitoring of transcription and translation in mammalian cell-free expression in bulk and in cell-sized droplets

et al. 2018

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Transcription and translation are two critical processes during eukaryotic gene expression that regulate cellular activities. The development of mammalian cell-free expression (CFE) systems provides a platform for studying these two critical processes in vitro for bottom-up synthetic biology applications such as construction of an artificial cell. Moreover, real-time monitoring of the dynamics of synthesized mRNA and protein is key to characterize and optimize gene circuits before implementing in living cells or in artificial cells. However, there are few tools for measurement of mRNA and protein dynamics in mammalian CFE systems. Here, we developed a locked nucleic acid (LNA) probe for monitoring transcription in a HeLa-based CFE system in real-time. By using this LNA probe in conjunction with a fluorescent reporter protein, we were able to simultaneously monitor mRNA and protein dynamics in bulk reactions and cell-sized single-emulsion droplets. We found rapid production of mRNA transcripts that decreased over time as protein production ensued in bulk reactions. Our results also showed that transcription in cell-sized droplets has different dynamics compared to the transcription in bulk reactions. The use of this LNA probe in conjunction with fluorescent proteins in HeLa-based mammalian CFE system provides a versatile in vitro platform for studying mRNA dynamics for bottom-up synthetic biology applications.

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Section: Introductionmentioning

confidence: 99%

Simultaneous monitoring of transcription and translation in mammalian cell-free expression in bulk and in cell-sized droplets

et al. 2018

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“…Recently, microfluidic technology has been used for the development of well-defined cell-sized lipid vesicles [38][39][40][41][42][43][44][45]. The growth of synthetic biology has enabled the expression, purification, and reconstitution of various protein types owing to the development of complex artificial cell models [46][47][48][49]. The giant lipid vesicles generated using microfluidic technology are monodispersive vesicles with high encapsulation efficiency.…”

Section: Introductionmentioning

confidence: 99%

Development of Artificial Cell Models Using Microfluidic Technology and Synthetic Biology

Kamiya

2020

Micromachines

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Giant lipid vesicles or liposomes are primarily composed of phospholipids and form a lipid bilayer structurally similar to that of the cell membrane. These vesicles, like living cells, are 5–100 μm in diameter and can be easily observed using an optical microscope. As their biophysical and biochemical properties are similar to those of the cell membrane, they serve as model cell membranes for the investigation of the biophysical or biochemical properties of the lipid bilayer, as well as its dynamics and structure. Investigation of membrane protein functions and enzyme reactions has revealed the presence of soluble or membrane proteins integrated in the giant lipid vesicles. Recent developments in microfluidic technologies and synthetic biology have enabled the development of well-defined artificial cell models with complex reactions based on the giant lipid vesicles. In this review, using microfluidics, the formations of giant lipid vesicles with asymmetric lipid membranes or complex structures have been described. Subsequently, the roles of these biomaterials in the creation of artificial cell models including nanopores, ion channels, and other membrane and soluble proteins have been discussed. Finally, the complex biological functions of giant lipid vesicles reconstituted with various types of biomolecules has been communicated. These complex artificial cell models contribute to the production of minimal cells or protocells for generating valuable or rare biomolecules and communicating between living cells and artificial cell models.

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“…Recombinant in vitro transcription-translation (TX-TL) procedures, also known as cell-free systems, have becoming increasingly common in recent years for a variety of biochemical and molecular procedures, mainly due to their versatility, and particularly in synthetic biology (Hammerling, Krüger, & Jewett, 2019;Keasling, 2012;Perez, Stark, & Jewett, 2016;Villarreal & Tan, 2017). These systems allow flexibility in the reaction condition of parameters for protein production, such as the transcription and translation machinery, as well as minimizing the possible limitations that carry working with living cells (Carlson, Gan, Hodgman, & Jewett, 2012;Silverman, Karim, & Jewett, 2019).…”

Section: Introductionmentioning

confidence: 99%

PURE mRNA display and cDNA display provide rapid detection of consensus binding motif via high-throughput sequencing

Reyes¹,

Kuruma²,

Fujimi³

et al. 2020

Preprint

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The recombinant in vitro translation system known as the PURE system has been used in a variety of cell-free experiments such as expression of native and de novo proteins as well as various display methods to select for functional polypeptides. We developed a refined PURE-based display method for the preparation of stable mRNA and cDNA-peptide conjugates and validated its utility for in vitro selection. Our conjugate formation efficiency exceeded 40%, followed by gel purification to allow minimum carry-over of components from the translation system to the downstream assay enabling clean and efficient random peptide sequence screening. As a proof-of-concept, we chose the commercially available anti-FLAG M2 antibody as a target molecule for validation. Starting from approximately 1.7 x 10 12 random sequences, a round-by-round high-throughput sequencing showed clear enrichment of the FLAG epitope DYKDDD as well as revealing consensus FLAG binding motif DYK(D/L/N)(L/Y/D/N/F)D. Enrichment of core FLAG motifs lacking one of the four key residues (DYKxxD) indicates that Tyr (Y) and Lys (K) appear as the two key residues essential for binding. Furthermore, comparison between mRNA display and cDNA display method resulted in overall similar performance with slightly higher enrichment for mRNA display. The consistency of two different display methods achieved by commercially available PURE system will be useful for future studies to explore sequence and functional space of diverse polypeptides.

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Cell-free systems in the new age of synthetic biology

Cited by 23 publications

References 80 publications

Simultaneous monitoring of transcription and translation in mammalian cell-free expression in bulk and in cell-sized droplets

Simultaneous monitoring of transcription and translation in mammalian cell-free expression in bulk and in cell-sized droplets

Development of Artificial Cell Models Using Microfluidic Technology and Synthetic Biology

PURE mRNA display and cDNA display provide rapid detection of consensus binding motif via high-throughput sequencing

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