Engineering site-specific recombinases for use in synthetic biology

Macfarlane, Hayley Louise (2017) Engineering site-specific recombinases for use in synthetic biology. PhD thesis, University of Glasgow.

Abstract

This project examined whether it was possible to create functional hybrid serine integrases – proteins responsible for recombining DNA in a site-specific manner. Creating hybrid recognition sites, specifically engineered to be recognised by the new integrases, was examined concurrently. Ultimately, new serine integrases and recognition sites were created with the intention of increasing the repertoire of serine integrases available for use as independently functioning modules in synthetic biology assemblies. Experiments were carried out primarily on two groups of hybrid integrases – BxbI integrase and ΦC31 integrase, and the smaller recombinase Tn3 resolvase and ΦC31 integrase.
It was determined that either the BxbI integrase/ΦC31 integrase hybrids were not active on hybrid or parental recognition sites, or that the proteins themselves were not expressed at a high enough level to exhibit any activity. However, one ΦC31 integrase/BxbI integrase hybrid did exhibit activity on ΦC31 integrase recognition sites in vivo, though not on hybrid sites.
However, Tn3 resolvase/ΦC31 integrase hybrid proteins proved far more promising. The two hybrids exhibited recombination on sites created for them, whilst exhibiting no activity on any parental recognition sites. When both Tn3 resolvase and either hybrid integrase were present in vitro, recombination on combination substrate plasmids containing one copy of the Tn3 resolvase recognition site res site I and one copy of a hybrid recognition site was much higher than for either hybrid against hybrid sites on its own.
Additionally, throughout this investigation, it was discovered that ΦC31 integrase cleaved and recombined several sites very dissimilar to its natural attP and attB sites.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Synthetic biology, genetics, molecular biology, biochemistry, site-specific recombination, recombinases, resolvases, Tn3 resolvase, ΦC31 integrase, phiC31 integrase, BxbI integrase, NM resolvase, serine integrases, serine recombinases, crystal structures, DNA-protein interactions, DNA, genetic engineering, gene editing.
• Subjects: Q Science > QH Natural history > QH301 Biology; Q Science > QH Natural history > QH345 Biochemistry; Q Science > QH Natural history > QH426 Genetics; Q Science > QR Microbiology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Funder's Name: Engineering and Physical Sciences Research Council (EPSRC)
• Supervisor's Name: Stark, Professor Marshall and Colloms, Dr. Sean
• Date of Award: 2017
• Embargo Date: 7 September 2020
• Depositing User: Dr H. L. Macfarlane
• Unique ID: glathesis:2017-8378
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 07 Sep 2017 09:03
• Last Modified: 11 Aug 2022 14:59
• Thesis DOI: 10.5525/gla.thesis.8378
• URI: https://theses.gla.ac.uk/id/eprint/8378