Engineering light-inducible transcription factors for control of gene expression

Royo Miguel, Sara 2024. Engineering light-inducible transcription factors for control of gene expression. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Light-oxygen-voltage (LOV) photoreceptors are ubiquitous blue light sensing domains that regulate the activity of various effectors, such as DNA binding domains. Illumination of a LOV domain results in the formation of a cysteinyl-flavin adduct between the protein and the chromophore. This signal is propagated to adjacent effectors and modulates their activity. LOV domains have been used for optogenetic tools, to control biological processes non-invasively with high spatiotemporal resolution. This thesis focuses on the study and engineering of LOV domain-based transcription factors for the development of optogenetic tools. Straightforward evaluation of function is necessary in protein engineering to assess novel constructs. Fluorescent RNA aptamers are RNA sequences that selectively bind and enhance the fluorescence of small molecules. They overcome some of the limitations of fluorescent proteins as reporter systems. In Chapter 2, the use of the Pepper aptamer as a reporter of transcription in optogenetic systems was explored. An improved synthesis of the fluorogen was established and photoinduced isomerisation was observed, deeming this system unsuitable to work in combination with LOV domain-based tools. The photoactivatable transcription factor EL222 has been extensively used to control gene expression by placing a gene after its cognate promoter. In Chapter 3, a novel approach was used to design EL222 chimeras with different DNA binding affinity that maintained photoactivation. EL222 was engineered to mimic the master biofilm regulator CsgD. One EL222 variant was able to bind the CsgD promoter naturally present in E. coli and induce biofilm formation under illuminated conditions. Rational design of optogenetic tools requires detailed knowledge of the signal transduction mechanism between adduct formation and effector activation. Seven variants of aureochrome LOV domain were analysed to understand the role of Asn194, hypothesised to be involved in the hydrogen bond network that links chromophore and effector. Changes in photocycle kinetics were recorded and the need for a hydrogen bond donor and acceptor amino acid in position 194 to maintain light induced dimerisation was identified. Additionally, three novel crystal structures of AuLOV variants were obtained.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Chemistry
Date of First Compliant Deposit:	4 February 2025
Last Modified:	04 Feb 2025 10:36
URI:	https://orca.cardiff.ac.uk/id/eprint/175875

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