Abstract
A major challenge facing bottom-up synthetic biology research is the construction ofprotocell populations able to respond to external signals, creating functional
protocellular ecosystems with emergent behaviours. The aim of this thesis is to
construct stimuli-responsive protocell models (proteinosomes) via chemical
programming of the crosslinked membrane as a step towards this.
The polyethylene glycol (PEG) based crosslinker traditionally used in
proteinosome synthesis has the potential to be easily functionalised with the addition
of responsive moieties. Chapter 2 presents the synthesis and characterisation of two
novel stimuli-responsive crosslinkers with embedded responsive moieties and
terminating N-hydroxy succinimide (NHS) groups for protein crosslinking. One cleaves
in response to UV light, and the other in response to a decrease in pH. The synthetic
routes and full characterisation are presented and crosslinker cleavage under a variety
of conditions is described.
The choice was made to focus research into light-responsive proteinosomes
due to ease of synthesis and higher potential with regards to disassembly control.
Chapter 3 outlines the synthesis and characterisation of chemically programmed lightresponsive proteinosomes. A UV laser in a confocal microscope system is employed
causing complete disassembly of the proteinosome membranes. Tuneable
parameters, laser power and laser scan speed, are investigated as methods to control
disassembly. Confocal software-controlled positioning systems allow targeting of
individual proteinosomes, and bespoke 2D patterning within proteinosome populations
in a simpler method not possible for other phenotypes of proteinosome.
Chapter 4 presents an in-depth analysis of the light-responsive proteinosome
membrane. A novel mathematical model to describe the inherent membrane pores is
produced, agreeing with experiments determining membrane molecular weight cutoff. An investigation light-induced selective release based on molecular weight of
substrates is shown with the permeability constants of substrates estimated. These
experiments are a step towards the design and synthesis of bespoke proteinosome
membranes and developing complex functional microsystems.
| Date of Award | 27 Sept 2022 |
|---|---|
| Original language | English |
| Awarding Institution |
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| Supervisor | Stephen Mann (Supervisor) |