Cardos Elena, Rosalía Paula;
(2021)
Towards the Development of a Genome Ablation Strategy for Synthetic Biology.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
A major goal of synthetic biology remains to create a fully orthogonal chassis, co-existing with its environment and unable to interfere with it, which would lead to the realisation of real-world applications of this discipline. Current biocontainment strategies often rely on easily circumvented auxotrophy or suicide networks triggered outside of a controlled environment. Rather than establishing a new genetic network to induce lethality, the present work intends to develop a method called genome ablation to obtain a DNA-free chassis unable to self-replicate or transfer genetic material to other species while remaining biochemically functional. In order to achieve absolute DNA degradation in vivo, exonuclease III and T5 bacteriophage exonuclease were overexpressed in Escherichia coli CSR603, a strain deficient in DNA repair, leading to DNA degradation without filamentation. Antibiotics mitomycin C and ciprofloxacin induced lethality and enhanced DNA degradation. Expression of restriction enzymes PvuII or HpaII, DNA gyrase poison ccdB, or T4 bacteriophage endonuclease denA alongside exonucleases reduced DNA content and cell viability; the lowest DNA content was achieved with E. coli CSR603 expressing PvuII at 4 h post-induction. Nuclease induction was found to exert a high selective pressure, leading to loss of nuclease activity. UV irradiation for 15 min induced lethality and absolute DNA degradation. However, this also induced extensive photodamage and compromised membrane integrity. Despite a functional chassis was not attained and further analysis is required, this is the first demonstration of DNA degradation in vivo applied to chassis engineering.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Towards the Development of a Genome Ablation Strategy for Synthetic Biology |
| Event: | UCL (University College London) |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2021. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices UCL > Provost and Vice Provost Offices > UCL BEAMS UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10122787 |
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