Recombinase-based cellular memory

: methods for reading and reliable writing as steps towards real-world applications

Abstract

Large serine recombinases (LSRs) recognise specific sites and catalyse irreversible structural changes in DNA such as inversions, excisions, or insertions. Hence, they are becoming popular tools for building DNA-based memory devices encoding writable digital information. Even complex memory circuits containing multiple registers, utilising multiple LSRs, have been created. However, methods to ‘read’ the information encoded in memory registers are often laboursome and slow, and leaky gene expression leads to malfunctions in these devices. To address these problems, in this thesis, we first developed NucleoBurst, an end-to-end workflow that can access the full state of thousands of memory registers of any size, in parallel, and at a nucleotide resolution, combining long-read nanopore sequencing, robotic lab automation, and bioinformatics analyses. We validated NucleoBurst by characterising a library of six LSRs. Then, we explored ways to stringently control gene expression in Escherichia coli. By simultaneously regulating transcription and translation, we show how basal expression of an inducible system can be reduced, with little impact on the maximum expression rate. Using this approach, we created several stringent expression systems harnessing multi-level regulation which can suppress transcriptional noise and create digital-like switches between ‘on’ and ‘off’ states. Finally, we deployed recombinase-based memory devices in plants and laid the basis for implementing NucleoBurst for memory readout in Arabidopsis thaliana roots. NucleoBurst’s semi-automated approach provides a means of quickly characterising the behaviour of DNA-based memory devices. This can allow us to rapidly expand the repertoire of characterised recombinases and observe the function of more complex systems built using them.
Our multi-level controllers illustrate the value of more diverse regulatory designs for synthetic biology and can enable the creation of tightly controlled devices. The experiments conducted on recombinase-based memory devices in plants can inform design principles for living memories and their effective use across a wider range of host organisms. An ability for cells to sense and potentially remember their own experiences could revolutionise many areas of biology and open up new avenues for synthetic biology.

Date of Award	3 Oct 2023
Original language	English
Awarding Institution	University of Bristol
Sponsors	The Royal Society
Supervisor	Claire S Grierson (Supervisor) & Thomas E Gorochowski (Supervisor)