Figure 5
In vitro evolution of circular DNA that can replicate in the presence of Cre recombinase. (a) Experimental scheme. (i) The transcription and translation-coupled DNA replication (TTcDR) reaction of the circular DNA was performed in a water-in-oil droplet in the presence of Cre recombinase. The average number of circular DNA molecules per droplet was adjusted to be less than one. In the TTcDR reaction, phi29 DNA polymerase was expressed and it synthesized a linear DNA using the circular DNA as a template. (ii) The synthesized DNA was recovered from the droplets, and (iii) circularized with a ligase after PCR amplification, during which mutations were induced. (iv) The circularized DNAs were re-encapsulated into new water-in-oil droplets containing the Cre recombinase and the TTcDR mixture. The concentration of Cre recombinase was determined based on the concentration of the DNA product in the previous round; the recombinase was increased or decreased when the DNA product increased or decreased, respectively. (b) Trajectories of the Cre recombinase concentration and the average concentration of the product DNA. The product DNA concentration was measured by qPCR after step (i). (c) The average replication ability of the DNA populations at the indicated rounds in the presence of Cre recombinase. The TTcDR reaction was performed with the circular DNA population (0.40 nM) at each round in the presence of 250 mU/μl Cre recombinase at 30 °C for 16 h. The error bars indicate standard deviation (n = 3).
