Combinatorial selection for replicable RNA by Qβ replicase while maintaining encoded gene function
The second selection cycle is the same as the first one (Fig 1) except for the changes in the in vivo selection method and omission of PCR amplification and mutagenesis. (1) A plasmid encoding the target gene, terminal recognition sites for Qβ replicase, and T7 promoter was transcribed in vitro with T7 RNA polymerase to produce an RNA. (2) In vitro RNA replication was performed with Qβ replicase. In this step, more replicable mutant RNAs from the library were selected. (3) The replicated RNAs were reverse transcribed into cDNAs and (4) ligated to a plasmid vector. (5) The ampicillin resistance plasmid was introduced into an E. coli strain with deleted target gene and complemented with a plasmid encoding sucB gene for negative selection. (6) After 10h incubation in the presence of ampicillin, the small colonies were transferred on a new agar plate containing 5% sucrose for selection of cells that lost the original complementary plasmid. An E. coli cell harboring the plasmid encoding the functional target gene would produce a large colony on this plate. (7) All large colonies were collected and plasmids extracted from the colony mixtures for the next cycle.