Engineering bacterial biosensors to explore the mammalian gut
File(s)
Author(s)
Robinson, Clare
Type
Thesis
Abstract
Bacterial whole-cell biosensors, bacteria genetically engineered to sense and respond to specific signals, are powerful tools particularly within the context of the gut. When combined with genetic circuits capable of maintaining a record of sensor activation, so called ‘memory’ circuits, these bacteria have the ability to non-invasively detect and report on the largely inaccessible gut environment. The identification of relevant biomarkers and development of associated sensing components is an identified bottleneck within the field, with the development of new whole-cell biosensors often being a time-consuming process with a high risk of failure when faced with the challenging gut environment. Here we optimise and extend the flexibility of a biosensor screening platform to simultaneously test hundreds of individually barcoded bacterial biosensor strains in vitro and directly in vivo in the murine gut. We investigate sensor design approaches, library construction, colonisation and diversity maintenance within the gut and optimise the analysis of sensor activation to build and test two new libraries of bacterial biosensors using two-component systems sourced from various gut bacteria or E. coli promoters responsive to various conditions, including inflammation. Through application of the screening pipeline, we construct, characterise, and validate 5 new bacterial biosensors (4 TCS-based sensors, 1 promoterbased sensor) responsive to physiologically relevant conditions within the gut, including a novel sensor found to be responsive to inflammation. We then design, build, and test a new fluorescent chassis that can be applied to characterise individual sensors and resolve spatial activation within the length of the gut, and demonstrate successful visualisation of sensor activation directly in the gut by microscopy. Our screening approach can be applied to any type of transcriptionally ctivated sensing systems, facilitating and accelerating new bacterial biosensor development that can be applied to further our understanding of the complex gut environment in health and disease.
Version
Open Access
Date Issued
2024-04-10
Date Awarded
01/10/2024
License URL
Advisor
Riglar, David
Sponsor
Imperial College London
Publisher Department
Department of Infectious Disease
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)