Development of a compartmentalised self-replication protocol for selection of superior blunt-end DNA ligases

Enzyme Microb Technol. 2023 Feb:163:110153. doi: 10.1016/j.enzmictec.2022.110153. Epub 2022 Nov 5.

Abstract

DNA ligases are widely used in molecular biology to generate recombinant DNA. However, having evolved for nick-sealing, they are inefficient at catalysing the blunt-ended ligations that are critical to many biotechnological applications, including next-generation sequencing. To facilitate engineering of superior blunt-ended DNA ligases, we have developed and validated a compartmentalised self-replication protocol that can select for the most effective ligases from a library of variants. Parallel cultures of Escherichia coli cells expressing different plasmid-encoded variants act as both a source of template DNA for discrete whole-plasmid PCR reactions, and a source of expressed ligase to circularise the corresponding PCR amplicons. The most efficient ligases generate the greatest number of self-encoding plasmids, and are thereby selected over successive rounds of transformation, amplification and ligation. By individually optimising critical steps, we arrived at a coherent protocol that, over five rounds of selection, consistently enriched for cells expressing the more efficient of two recombinant DNA ligases.

Keywords: Compartmentalized selfreplication; Competitive selection; DNA ligase; Enzyme engineering.

MeSH terms

  • DNA Ligases* / genetics
  • DNA, Recombinant*
  • Escherichia coli / genetics
  • Ligases / genetics
  • Plasmids / genetics
  • Polymerase Chain Reaction

Substances

  • DNA Ligases
  • DNA, Recombinant
  • Ligases