The development of an orthogonal DNA replication system comprised of a ‘replisome plasmid’ (RP, maintained by the host) and an orthogonal ‘episome plasmid’ (EP, maintained by the RP, not the host) in E. coli allows for major repurposing of the exogenous replisome’s mutation rate beyond the host’s genomic error threshold for continuous evolution of genes of interest. Orthogonal replication in E. coli has been described for several lytic coliphages that encode their own replisome. However, such phages kill the host upon infection and thus do not require mechanisms to maintain their copy number at a level sustainable for the host organism. In contrast, lysogenic phages have evolved strategies to control the copy number of their genomes after infection to achieve prolonged lysogeny with their host, however these phages rely on the host replication machinery. My strategy for developing an orthogonal replication system in E. coli is therefore based upon an RP based on the orthogonal replisome of a lytic phage (bacteriophage T7) and an EP based on the control mechanisms of a lysogenic phage (bacteriophage N15). T7 replication involves five essential proteins: T7 DNA polymerase, DNA helicase/primase, the ssDNA binding protein, T7 RNA polymerase, and the host factor E. coli thioredoxin. I have designed an RP comprising the T7 replisome. To confirm that all genes are functional, E. coli. strains that had been transformed with the RP have been infected with T7 phages in which the four essential phage replication genes had been knocked out. In all cases, phage growth was recovered, demonstrating that the expressed replication proteins are functional. The T7 replisome is highly processive and replication of circular plasmid DNA leads to formation of toxic concatemeric DNA through rolling circle replication. To avoid this, I chose a linear EP based on a lysogenic N15 phage vector. The N15 protelomerase TelN cuts DNA at specific recognition sequences at both ends of the linear plasmid to form covalently closed ends at the cleavage site, thus preventing formation of high molecular weight DNA by rolling circle replication I have cloned the EP with the T7 origin of replication, as well as the N15 protelomerase. Maintenance of the EP by the RP has been confirmed. Mutations of T7 DNA polymerase mutants lacking its proofreading exonuclease activity, as well as additional mutations in the palm domain are investigated for increasing the mutation rate of the orthogonal replisome. ]

An orthogonal replication system for continuous evolution of proteins in E. coli

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