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Engineering AraC to make it responsive to light instead of arabinose

Romano, Edoardo

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Engineering methods to spatiotemporally control gene expression is one of the most important goals of synthetic biology. While some level of temporal and spatial control of gene expression can be achieved with chemicals added to the cell population, sophisticated regulation cannot be achieved in this way. The rapidly emerging field of optogenetics offers the solution to this problem because the light is a perfect spatially and temporally confinable external trigger, enabling the easy regulation of the gene expression process in individual cells within a population or layer of cells. Here, I present the engineering of the natural L-arabinose-responsive bacterial transcriptional activator AraC in the non-photoresponsive bacterium Escherichia coli to render it light-inducible. Several optogenetic systems that regulate transcription in bacteria have already been developed. With my work, I aim to provide the scientific community with a tool for easily switching the induction trigger from L-arabinose to light in pre-existing L-arabinose-responsive plasmids and strains. AraC activates transcription from the PBAD promoter through dimer rearrangement after binding of L-arabinose. The dimerization domain was swapped with the blue light-triggered dimerizing protein VVD from Neurospora crassa to drive AraC dimerization with light and, consequently, control its ability to activate PBAD. Initially, a small library of fusion constructs was created, whose expression was dependent on IPTG induction and the inducible promoter was cloned in a different plasmid. I further engineered the system, removing the IPTG dependence for VVD-AraC expression and cloning the transcription factor in the same plasmid as the PBAD and the reporter. Then I optimized the induction protocol and enlarged the initial library, obtaining higher and more reproducible induction levels. I characterized this small family of novel blue light-inducible AraC dimers in E. coli, named BLADE, to finely control gene expression in space and time. I compared BLADE with wild-typeAraC in terms of inducer catabolism, induction reversibility and population heterogeneity, highlighting the strengths and weaknesses of each. To showcase BLADE’s ability to spatiotemporally control gene expression, I performed bacteriography, a method that relies on the selective passage of light through a photomask to reproduce, using bacteria, complex images. Among others, I reproduced the Blade Runner movie poster and Michelangelo’s “Creation of Adam” fresco with unprecedented quality. I also investigated the mechanism of BLADE action in vivo, showing the formation of aggregates in the dark, which I speculate contribute to the tightness of the system. I successfully demonstrated the applicability of BLADE in inducing with light gene expression from plasmids and strains that normally would respond to L-arabinose. These results prove that BLADE enables optogenetic experiments to be done with pre-existing L-arabinose-inducible systems without the need to clone, a distinctive feature that no other light-inducible system has. I employed BLADE to regulate the expression of proteins involved in cell division (MinD and its mutant MinD10) and cell shape (MreB and RodZ), showing that light can be applied to control bacterial cell morphology, which paves the way to more sophisticated studies of the effect of environmental factors on morphology in the future. Using BLADE to overexpress MinD10, I demonstrate that minicell formation can be triggered at a specific time point, which offers the possibility to obtain minicells of similar metabolic activity, that bears potential benefits for the use of minicells as delivery vehicles. To showcase the advantage of light as an external trigger in medium7 and high-throughput assays, I built a library of 117 constructs to characterize 39 E. coli genes with unknown or poorly defined function in terms of intracellular localization and effect on cell growth and morphology of their overexpression. I identified several proteins that, when overexpressed, affect cell growth, both positively and negatively; I also found two proteins whose overexpression leads to cell elongation and another one that exhibits a toxic effect. Lastly, through fusion to a fluorescent reporter, I determined their localization. In conclusion, I believe that BLADE is a robust and effective optogenetic tool for the study of bacterial gene regulatory networks and gene function. I expect that its plug-and-play functionality, together with its tight induction control and its reliable performances, will allow its adoption in microbiology, synthetic biology and biotechnology.

Document type: Dissertation
Supervisor: Di Ventura, Prof. Dr. Barbara
Place of Publication: Heidelberg
Date of thesis defense: 14 May 2021
Date Deposited: 22 Nov 2021 12:35
Date: 2021
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Service facilities > Bioquant
DDC-classification: 000 Generalities, Science
570 Life sciences
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