Open Access Te Herenga Waka-Victoria University of Wellington
Browse
thesis_access.pdf (3.29 MB)

Evolution and Evaluation of Engineered DNA Ligases for Improved Blunt-End Ligation

Download (3.29 MB)
Version 2 2023-09-26, 23:59
Version 1 2021-12-08, 20:08
thesis
posted on 2023-09-26, 23:59 authored by Sharma, Janine

DNA ligases are fundamental enzymes in molecular biology and biotechnology where they perform essential reactions, e.g. to create recombinant DNA and for adaptor attachment in next-generation sequencing. T4 DNA ligase is the most widely used commercial ligase owing to its ability to catalyse ligation of blunt-ended DNA termini. However, even for T4 DNA ligase, blunt-end ligation is an inefficient activity compared to cohesive-end ligation, or its evolved activity of sealing single-strand nicks in double-stranded DNA. Previous research from Dr Wayne Patrick showed that fusion of T4 DNA ligase to a DNA-binding domain increases the enzyme’s affinity for DNA substrates, resulting in improved ligation efficiency. It was further shown that changes to the linker region between the ligase and DNA-binding domain resulted in altered ligation activity. To assist in optimising this relationship, we designed a competitive ligase selection protocol to enrich for engineered ligase variants with greater blunt-end ligation activity. This selection involves expressing a DNA ligase from its plasmid construct, and ligating a linear form of its plasmid, sealing a double-strand DNA break in the chloramphenicol resistance gene, permitting bacterial growth. Previous researcher Dr Katherine Robins created two linker libraries of 33 and 37 variants, from lead candidate ligase-cTF and (the less active form of p50-ligase variant) ligase-p50, respectively. Five rounds of selection were applied to each library. One linker variant, denoted ligase-CA3 showed the greatest improvement, comprising 42% of the final selected ligase-cTF population. In contrast, a lead linker variant from the ligase-p50 library was not obtained. In this study one additional round of selection was applied to the ligase-p50 library to test whether a lead variant would emerge. However, the linker variants selected at the end of Round 6 did not suggest a clear lead candidate, so one of the top variants (ligase-PPA17) was selected to represent this population in a fluorescence-based ligation assay that I optimised. Following identification of optimal reaction buffers to improve protein stability and DNA ligation, six engineered variants were compared for blunt-, cohesive-end, and nick sealing ligation activities. All five engineered variants exhibited improved blunt-end ligation activity over T4 DNA ligase. Ligase-PPA17 (1.9-fold improvement over T4 DNA ligase) was best performing for blunt-end ligation. This study found no evidence that ligase-CA3 was significantly improved over its predecessor, ligase-cTF in blunt-end ligation, however it was the best performing variant at cohesive-end ligation. Overall, we have evolved DNA ligase variants with improved blunt-end ligation activity over T4 DNA ligase which may be more advantageous in molecular biology and biotechnology for a variety of applications.

History

Copyright Date

2020-01-01

Date of Award

2020-01-01

Publisher

Te Herenga Waka—Victoria University of Wellington

Rights License

CC BY-NC-ND 4.0

Degree Discipline

Cell and Molecular Bioscience

Degree Grantor

Te Herenga Waka—Victoria University of Wellington

Degree Level

Masters

Degree Name

Master of Science

Victoria University of Wellington Unit

Centre for Biodiscovery

ANZSRC Type Of Activity code

4 EXPERIMENTAL DEVELOPMENT

Victoria University of Wellington Item Type

Awarded Research Masters Thesis

Language

en_NZ

Victoria University of Wellington School

School of Biological Sciences

Advisors

Ackerley, David; Patrick, Wayne