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      Recent advances of droplet-based microfluidics for engineering artificial cells

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      SLAS Technology
      Elsevier BV

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          Most cited references78

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          Creating, transporting, cutting, and merging liquid droplets by electrowetting-based actuation for digital microfluidic circuits

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            Fluorescence-activated droplet sorting (FADS): efficient microfluidic cell sorting based on enzymatic activity.

            We describe a highly efficient microfluidic fluorescence-activated droplet sorter (FADS) combining many of the advantages of microtitre-plate screening and traditional fluorescence-activated cell sorting (FACS). Single cells are compartmentalized in emulsion droplets, which can be sorted using dielectrophoresis in a fluorescence-activated manner (as in FACS) at rates up to 2000 droplets s(-1). To validate the system, mixtures of E. coli cells, expressing either the reporter enzyme beta-galactosidase or an inactive variant, were compartmentalized with a fluorogenic substrate and sorted at rates of approximately 300 droplets s(-1). The false positive error rate of the sorter at this throughput was <1 in 10(4) droplets. Analysis of the sorted cells revealed that the primary limit to enrichment was the co-encapsulation of E. coli cells, not sorting errors: a theoretical model based on the Poisson distribution accurately predicted the observed enrichment values using the starting cell density (cells per droplet) and the ratio of active to inactive cells. When the cells were encapsulated at low density ( approximately 1 cell for every 50 droplets), sorting was very efficient and all of the recovered cells were the active strain. In addition, single active droplets were sorted and cells were successfully recovered.
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              The All E. coli TX-TL Toolbox 2.0: A Platform for Cell-Free Synthetic Biology.

              We report on and provide a detailed characterization of the performance and properties of a recently developed, all Escherichia coli, cell-free transcription and translation system. Gene expression is entirely based on the endogenous translation components and transcription machinery provided by an E. coli cytoplasmic extract, thus expanding the repertoire of regulatory parts to hundreds of elements. We use a powerful metabolism for ATP regeneration to achieve more than 2 mg/mL of protein synthesis in batch mode reactions, and more than 6 mg/mL in semicontinuous mode. While the strength of cell-free expression is increased by a factor of 3 on average, the output signal of simple gene circuits and the synthesis of entire bacteriophages are increased by orders of magnitude compared to previous results. Messenger RNAs and protein degradation, respectively tuned using E. coli MazF interferase and ClpXP AAA+ proteases, are characterized over a much wider range of rates than the first version of the cell-free toolbox. This system is a highly versatile cell-free platform to construct complex biological systems through the execution of DNA programs composed of synthetic and natural bacterial regulatory parts.
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                Author and article information

                Contributors
                Journal
                SLAS Technology
                SLAS Technology
                Elsevier BV
                24726303
                May 2023
                May 2023
                Article
                10.1016/j.slast.2023.05.002
                3f6072d3-1679-44bf-8760-2ca5e7131393
                © 2023

                https://www.elsevier.com/tdm/userlicense/1.0/

                http://creativecommons.org/licenses/by-nc-nd/4.0/

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