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.
The appearance of molecular replicators (molecules that can be copied) was probably a critical step in the origin of life. However, parasitic replicators take over, and would have prevented life from taking off, unless the replicators were compartmentalized in reproducing protocells. Paradoxically, control of protocell reproduction would seem to require evolved replicators. We show here that a simpler population structure, based on cycles of transient compartmentalization (TC) and mixing of RNA replicators, is sufficient to prevent takeover by parasitic mutants. TC tends to select for ensembles of replicators that replicate at a similar rate, including a diversity of parasites that could serve as a source of opportunistic functionality. Thus TC in natural, non-biological compartments could have allowed life to take hold. Main text:The earliest molecular replicators (1, 2) must have been plagued by freeloading parasitic replicators (3-6).For example, when the RNA genome of the Q virus was replicated in vitro using the viral replicase, 83% of the genome was deleted due to selection for RNAs with the fastest replication rate (7). Eventually, reproducing compartments (protocells) must have arisen, taming parasites by spatially limiting their propagation and allowing group selection at the compartment level, preventing functional collapse (5,(8)(9)(10). Indeed, serial fusion-division cycles of water-in-oil emulsion
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