Abstract
Automation is a useful strategy to make laborious evolutionary experiments faster and easier. To date, several types of continuous flow reactors have been developed for the automated evolutionary experiments of viruses and bacteria. However, the development of a flow reactor applicable to compartmentalized in vitro self-replication systems is still a challenge. In this study, we demonstrate automated in vitro evolution of a translation-coupled RNA system in a droplet flow reactor for the first time. This reactor contains approximately 1010 micro-scale droplets (average diameter is approximately 0.8 μm), which continuously fuse and divide among each other at a controllable rate. In the droplets, an RNA (artificial genomic RNA) replicate through the translation of self-encoded RNA replicase with spontaneously appearing parasitic RNA. We performed two automated replication experiments for more than 400 hours with different mixing intensities. We found that several mutations displayed increased frequencies in the genomic RNA populations and the dominant RNA mutants acquired the ability to replicate faster or acquired resistance to the parasitic RNA, demonstrating that Darwinian evolution occurred during the long-term replication. The droplet flow reactor we developed can be a useful tool to perform in vitro evolutionary experiments of translation-coupled systems.
Highlights
One useful method to understand complicated biological phenomena is to reconstitute these systems from a set of molecules in vitro
The translation-coupled RNA replication system used in this study consists of an artificial genomic RNA and a reconstituted translation system of E. coli
The genomic RNA encodes the catalytic subunit of an RNA replicase (Qbeta replicase), which is translated and forms an active replicase with EF-Tu and EF-Ts in the translation system
Summary
One useful method to understand complicated biological phenomena is to reconstitute these systems from a set of molecules in vitro. A similar evolutionary process was simulated in vitro by Spiegelman’s group in 196718 They repeated the replication of a genomic RNA of the bacteriophage Qbeta in the presence of RNA replicase through a long serial transfer experiment and observed the evolution of smaller RNAs. Later, other self-replication systems of RNA or DNA were constructed and evolution was observed in some cases[19,20,21]. One of the largest hurdles in performing evolution experiments is the requirement of laborious experimental processes for long durations To overcome this obstacle, automated systems, such as continuous flow stirred tank reactors (CSTRs) or chemostats, have been developed to examine the experimental evolution of bacteriophages[26,27,28], bacteria[29,30,31], fungi[32], and artificial self-replicating DNAs33. The duration of the RNA replication was not sufficient to observe evolution
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