Artificial genomic DNA replication and evolution outside of the cell (IMAGE)
Caption
This system consists of a circular DNA and a customized reconstituted cell-free gene expression system of E. coli, including T7 RNA polymerase and dNTP. The circular DNA contains the phi29 DNA polymerase and Cre recombinase genes under the T7 promoter and loxP site for recombination by Cre recombinase. First, phi29 DNA polymerase and Cre recombinase are expressed through transcription and translation. Next, the expressed phi29 DNA polymerase initiates rolling-circle replication to produce a long single-stranded DNA, then synthesizes the complementary strand to produce long double-stranded DNA. Finally, the translated Cre recombinase catalyzes homologous recombination at loxP sites to reproduce circular DNA. When encapsulated this system into micro-scale droplets and continue the replication for many generations through serial dilution cycle by supplying the cell-free gene expression system, the DNA accumulate mutations to replicate more via Darwinian evolution.
Credit
© 2004 American Chemical Society
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