Sealable Femtoliter Chamber Arrays for Cell-free Biology

Norred, S. Elizabeth; Caveney, Patrick M.; Retterer, Scott T.; Boreyko, Jonathan B.; Fowlkes, Jason D.; Collier, C. Patrick; Simpson, Michael L.

doi:10.3791/52616

Cited by 6 publications

(13 citation statements)

References 49 publications

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“…Conversely, in vitro reaction chambers are especially suited for isolating the effects of specific mechanisms from confounding cellular processes, − and cell-free protein synthesis (CFPS) systems have been successfully used to observe gene expression bursting . Recently, arrays of microfabricated cellular-scale reaction chambers have been demonstrated to be a viable way to confine CFPS reactions to study gene expression, in particular the noise in expression. , Bursting and noise are inseparably linked as bursting is often the dominant contributor to expression noise, ,,,, and noise measurements are often used to understand the underlying dynamics of gene expression in vivo . ,,, In combination, microfabricated cell-scale reactors and gene expression noise measurements provide a unique platform to explore gene expression bursting and resource sharing in well-controlled and easily manipulated environments.…”

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confidence: 99%

Resource Sharing Controls Gene Expression Bursting

et al. 2016

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Episodic gene expression, with periods of high expression separated by periods of no expression, is a pervasive biological phenomenon. This bursty pattern of expression draws from a finite reservoir of expression machinery in a highly time variant way, i.e., requiring no resources most of the time but drawing heavily on them during short intense bursts, that intimately links expression bursting and resource sharing. Yet, most recent investigations have focused on specific molecular mechanisms intrinsic to the bursty behavior of individual genes, while little is known about the interplay between resource sharing and global expression bursting behavior. Here, we confine Escherichia coli cell extract in both cell-sized microfluidic chambers and lipid-based vesicles to explore how resource sharing influences expression bursting. Interestingly, expression burst size, but not burst frequency, is highly sensitive to the size of the shared transcription and translation resource pools. The intriguing implication of these results is that expression bursts are more readily amplified than initiated, suggesting that burst formation occurs through positive feedback or cooperativity. When extrapolated to prokaryotic cells, these results suggest that large translational bursts may be correlated with large transcriptional bursts. This correlation is supported by recently reported transcription and translation bursting studies in E. coli. The results reported here demonstrate a strong intimate link between global expression burst patterns and resource sharing, and they suggest that bursting plays an important role in optimizing the use of limited, shared expression resources.

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confidence: 99%

Resource Sharing Controls Gene Expression Bursting

et al. 2016

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“…. The implication is that dilution of expression resources changed expression in a way that inverted the relationship between the mean and the variance of YFP concentration, which is most often modeled as 8,31,35,36…”

Section: Resultsmentioning

confidence: 99%

Self-organization controls expression more than abundance of molecular components of transcription and translation in confined cell-free gene expression

Caveney¹,

Dabbs²,

Chauhan³

et al. 2018

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Cell-free gene expression using purified components or cell extracts has become an important platform for synthetic biology that is finding a growing number of practical applications. Unfortunately, at cell-relevant reactor volumes, cell-free expression suffers from excessive variability (noise) such that protein concentrations may vary by more than an order of magnitude across a population of identically constructed reaction chambers. Consensus opinion holds that variability in expression is due to the stochastic distribution of expression resources (DNA, RNAP, ribosomes, etc.) across the population of reaction chambers. In contrast, here we find that chamber-to-chamber variation in the expression efficiency generates the large variability in protein production. Through analysis and modeling, we show that chambers self-organize into expression centers that control expression efficiency. Chambers that organize into many centers, each having relatively few expression resources, exhibit high expression efficiency. Conversely, chambers that organize into just a few centers where each center has an abundance of resources, exhibit low expression efficiency. A particularly surprising finding is that diluting expression resources reduces the chamber-to-chamber variation in protein production. Chambers with dilute pools of expression resources exhibit higher expression efficiency and lower expression noise than those with more concentrated expression resources. In addition to demonstrating the means to tune expression noise, these results demonstrate that in cell-free systems, self-organization may exert even more influence over expression than the abundance of the molecular components of transcription and translation. These observations in cell-free platform may elucidate how self-organized, membrane-less structures emerge and function in cells.

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“…Successful synthesis of different antibody formats, including single-chain variable fragments (scFvs), Fab fragments, as well as complete IgGs, has already been shown in E. coli [114,115], Sf21 [10,116], reticulocyte [110], wheat germ [117], and CHO CF systems [46,75,118]. Furthermore, the upscaling of CF reactions to the liter-scale [25,115] as well as downscaling [119] and high-throughput applications [120] have been demonstrated.…”

Section: Antibody Productionmentioning

confidence: 99%

Cell-Free Protein Synthesis: A Promising Option for Future Drug Development

et al. 2020

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Proteins are the main source of drug targets and some of them possess therapeutic potential themselves. Among them, membrane proteins constitute approximately 50% of the major drug targets. In the drug discovery pipeline, rapid methods for producing different classes of proteins in a simple manner with high quality are important for structural and functional analysis. Cell-free systems are emerging as an attractive alternative for the production of proteins due to their flexible nature without any cell membrane constraints. In a bioproduction context, open systems based on cell lysates derived from different sources, and with batch-to-batch consistency, have acted as a catalyst for cell-free synthesis of target proteins. Most importantly, proteins can be processed for downstream applications like purification and functional analysis without the necessity of transfection, selection, and expansion of clones. In the last 5 years, there has been an increased availability of new cell-free lysates derived from multiple organisms, and their use for the synthesis of a diverse range of proteins. Despite this progress, major challenges still exist in terms of scalability, cost effectiveness, protein folding, and functionality. In this review, we present an overview of different cell-free systems derived from diverse sources and their application in the production of a wide spectrum of proteins. Further, this article discusses some recent progress in cell-free systems derived from Chinese hamster ovary and Sf21 lysates containing endogenous translocationally active microsomes for the synthesis of membrane proteins. We particularly highlight the usage of internal ribosomal entry site sequences for more efficient protein production, and also the significance of site-specific incorporation of non-canonical amino acids for labeling applications and creation of antibody drug conjugates using cell-free systems. We also discuss strategies to overcome the major challenges involved in commercializing cell-free platforms from a laboratory level for future drug development. Key PointsCell-free protein synthesis has the potential to become an alternative method for the rapid and highly parallel expression of a diverse range of proteins.Cell-free systems utilize the translation machinery of the cells, bypassing the constraints of the cell membrane and thus offer openness and configurational flexibility even for the synthesis of difficult-to-express proteins.In comparison to traditional approaches, cell-free systems can be time-saving, from initial synthesis to downstream applications.

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Sealable Femtoliter Chamber Arrays for Cell-free Biology

Cited by 6 publications

References 49 publications

Resource Sharing Controls Gene Expression Bursting

Resource Sharing Controls Gene Expression Bursting

Self-organization controls expression more than abundance of molecular components of transcription and translation in confined cell-free gene expression

Cell-Free Protein Synthesis: A Promising Option for Future Drug Development

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