Engineering a genetic circuit for Turing patterns in E. coli with a Synthetic Biology approach
File(s)
Supplementary information
Author(s)
Tica, Jure
Type
Thesis
Abstract
Genetic circuits that can form spatial patterns have been a major topic of interest within Synthetic Biology. Turing patterns are self-organising spatial wave, spot or labyrinthine patterns that are formed in some reaction-diffusion circuits. The simplest Turing circuit involves a slow-diffusing activator and a fast-diffusing inhibitor, interacting to regulate their own and each other’s rates of production. An unambiguous implementation of Turing patterns with a genetic circuit is still lacking because of their exquisitely fine-tuned nature. This study aims to address this shortcoming and sets out to engineer a genetic circuit for Turing patterning in E. coli from first principles.
Two genetic circuits were studied. Firstly, a phage circuit was designed according to the minimal self-activation, lateral inhibition Turing topology and involves a slow-diffusing M13 filamentous phage and a fast-diffusing 3OC6HSL quorum sensing signal. This circuit was abandoned because of the many complexities of phage biology, which were working against its successful implementation as a Turing generator. The focus was shifted to circuit ‘3954’, which was designed according to a more robust three-node topology and implemented with two small molecule diffusors; this could be done because the circuit allows for equal diffusivity of the two diffusing signals. All the components of circuit ‘3954’ were tested in reduced subcircuits and were shown to be functioning as expected. Growing bacterial colonies bearing the circuit were then visualised for pattern formation using confocal microscopy. Even though no Turing patterns were detected, the colonies consistently showed a centre-surround expression pattern of the fluorescence reporters, where GFP was expressed at the colony centre, whereas mCherry was predominantly expressed at the periphery. The obtained reaction-diffusion patterns are a good foundation for further tuning and exploration.
Two genetic circuits were studied. Firstly, a phage circuit was designed according to the minimal self-activation, lateral inhibition Turing topology and involves a slow-diffusing M13 filamentous phage and a fast-diffusing 3OC6HSL quorum sensing signal. This circuit was abandoned because of the many complexities of phage biology, which were working against its successful implementation as a Turing generator. The focus was shifted to circuit ‘3954’, which was designed according to a more robust three-node topology and implemented with two small molecule diffusors; this could be done because the circuit allows for equal diffusivity of the two diffusing signals. All the components of circuit ‘3954’ were tested in reduced subcircuits and were shown to be functioning as expected. Growing bacterial colonies bearing the circuit were then visualised for pattern formation using confocal microscopy. Even though no Turing patterns were detected, the colonies consistently showed a centre-surround expression pattern of the fluorescence reporters, where GFP was expressed at the colony centre, whereas mCherry was predominantly expressed at the periphery. The obtained reaction-diffusion patterns are a good foundation for further tuning and exploration.
Version
Open Access
Date Issued
2020-11
Date Awarded
2021-04
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
Isalan, Mark
Sponsor
Volkswagen Foundation (Firm)
Grant Number
LIFE: 93 065
Publisher Department
Life Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)