Chemical and pore-mediated programming of in vitro transcription–translation in droplet-interface-bilayer artificial cells

Colborne, Matthew 2025. Chemical and pore-mediated programming of in vitro transcription–translation in droplet-interface-bilayer artificial cells. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Bottom-up synthetic biology aims to construct cell-like systems, artificial cells, from defined molecular components. While individual behaviours such as membrane compartmentalisation, protein expression, and programmability have each been demonstrated, integrating them within a single platform remains a challenge. Droplet interface bilayers (DIBs) provide a modular artificial cell chassis through tunable membrane composition and addressable aqueous compartments. This thesis develops stable compartmentalisation, communication, and programmable expression within a single DIB-based system. A stabilisation workflow was established supporting in vitro transcription–translation (IVTT) inside DIBs for over 24 hours, the operational lifetime required for multi-step reactions. Systematic tuning of droplet and lipid compositions defined reproducible conditions for both PURExpress and S30 systems. To enable communication, pore-forming proteins were characterised as engineered membrane gates. α-Haemolysin served as a benchmark for small-molecule exchange and single-channel characterisation. Perfringolysin O (PFO), a cholesterol-dependent cytolysin, was developed for macromolecular transport. Membrane composition was screened to balance the cholesterol requirement for PFO assembly against its destabilising effect on bilayer mechanics. The resulting formulation enabled the first reported demonstration of macromolecular transfer in DIBs, extending communication beyond the small-molecule limit of existing pores. Programmability was developed by manipulating the reaction state of IVTT, bypassing the need for bespoke genetic regulation. The initial depletion of the nucleoside triphosphates identified small molecules as a target governing reversibility. By targeting these, a stable, reversible OFF state was achieved, and replenishment during assembly enabled switch-like behaviour. In DIB networks, their pore-mediated delivery from sender to receiver droplets activated expression remotely, producing temporally and spatially defined outputs. Together, these contributions establish a layered framework for stable compartmentalisation, pore-mediated communication, and programmable expression in DIB artificial cells. By integrating capabilities previously developed in isolation, the work extends the toolkit for engineering artificial cells that are stable, responsive, and programmable, with applications in deployable biosensing, controlled therapeutic delivery, and distributed cell-free biomanufacturing.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Schools > Pharmacy
Subjects:	Q Science > Q Science (General)
Funders:	EU Horizon - Artificial Cells with Distributed Cores (AC/DC)
Date of First Compliant Deposit:	28 October 2026
Last Modified:	28 Oct 2026 15:21
URI:	https://orca.cardiff.ac.uk/id/eprint/190503

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