Comparison of the burden and basal performance of optogenetic expression systems

Abstract

Optogenetic circuits offer the unique capabilities of fully reversible and light-tuneable modulation, facilitating precise spatiotemporal control of gene expression. Yet, light-inducible vectors are not broadly implemented in industry and research. Compared to popular chemical induction systems, optogenetic circuits have inherently high basal expression levels. Basal expression is a concern for all synthetic vectors as leaky gene expression during early growth phases can destabilise the bacterial host. The redirection of resources toward recombinant processes impedes host metabolism and slows growth. Comparative research into the basal performance and robustness of optogenetic expression vectors is relatively uncommon. Yet, comparative studies and characterisation of optogenetic circuits in different host strains would expand the usability of optogenetic control. The vectors pDusk and pDawn are constitutively expressed and gene expression is repressed or induced by blue light. Alternatively, the two-plasmid OptoT7RNAP system induces gene transcription via a photo-dimerizing T7 RNA polymerase. In this system, the photoactivable T7 RNA polymerase and gene of interest are transcribed from sperate plasmids. In this work, I compared the key circuit components that improved basal performance, reduced burden, and highlighted the strategies that may aid the broader implementation of optogenetics in the future. To compare the basal performance of pDusk, pDawn and OptoT7RNAP, I used high-throughput cell-culturing of E. coli transformants in microplates. Fluorescent reporter proteins were used for fast and efficient analysis of basal gene expression and optical density measurements were used to monitor bacterial proliferation over time. To characterise the metabolic burden associated with plasmid maintenance, replication and basal gene expression, population models were used to estimate bacterial growth properties. I found that photo-dimerizing T7RNAP modulation of gene expression greatly improved basal performance but the expression of a two plasmid system increased the doubling time of the E. coli host by 90%.

Date of Award	24 Jan 2023
Original language	English
Awarding Institution	University of Bristol
Supervisor	Thomas E Gorochowski (Supervisor) & Sabine Hauert (Supervisor)