<> "The repository administrator has not yet configured an RDF license."^^ . <> . . "Engineering AraC to make it responsive to light instead of arabinose"^^ . "Engineering methods to spatiotemporally control gene expression is one of the most important goals\r\nof synthetic biology. While some level of temporal and spatial control of gene expression can be\r\nachieved with chemicals added to the cell population, sophisticated regulation cannot be achieved in\r\nthis way. The rapidly emerging field of optogenetics offers the solution to this problem because the\r\nlight is a perfect spatially and temporally confinable external trigger, enabling the easy regulation of\r\nthe gene expression process in individual cells within a population or layer of cells.\r\nHere, I present the engineering of the natural L-arabinose-responsive bacterial transcriptional\r\nactivator AraC in the non-photoresponsive bacterium Escherichia coli to render it light-inducible.\r\nSeveral optogenetic systems that regulate transcription in bacteria have already been developed. With\r\nmy work, I aim to provide the scientific community with a tool for easily switching the induction\r\ntrigger from L-arabinose to light in pre-existing L-arabinose-responsive plasmids and strains.\r\nAraC activates transcription from the PBAD promoter through dimer rearrangement after binding of\r\nL-arabinose. The dimerization domain was swapped with the blue light-triggered dimerizing protein\r\nVVD from Neurospora crassa to drive AraC dimerization with light and, consequently, control its\r\nability to activate PBAD. Initially, a small library of fusion constructs was created, whose expression\r\nwas dependent on IPTG induction and the inducible promoter was cloned in a different plasmid.\r\nI further engineered the system, removing the IPTG dependence for VVD-AraC expression and\r\ncloning the transcription factor in the same plasmid as the PBAD and the reporter. Then I optimized\r\nthe induction protocol and enlarged the initial library, obtaining higher and more reproducible\r\ninduction levels.\r\nI characterized this small family of novel blue light-inducible AraC dimers in E. coli, named\r\nBLADE, 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,\r\nhighlighting the strengths and weaknesses of each. To showcase BLADE’s ability to spatiotemporally\r\ncontrol gene expression, I performed bacteriography, a method that relies on the selective passage of\r\nlight through a photomask to reproduce, using bacteria, complex images. Among others, I reproduced\r\nthe Blade Runner movie poster and Michelangelo’s “Creation of Adam” fresco with unprecedented\r\nquality. I also investigated the mechanism of BLADE action in vivo, showing the formation of\r\naggregates in the dark, which I speculate contribute to the tightness of the system.\r\nI successfully demonstrated the applicability of BLADE in inducing with light gene expression from\r\nplasmids and strains that normally would respond to L-arabinose. These results prove that BLADE\r\nenables optogenetic experiments to be done with pre-existing L-arabinose-inducible systems without\r\nthe need to clone, a distinctive feature that no other light-inducible system has.\r\nI employed BLADE to regulate the expression of proteins involved in cell division (MinD and its\r\nmutant MinD10) and cell shape (MreB and RodZ), showing that light can be applied to control\r\nbacterial cell morphology, which paves the way to more sophisticated studies of the effect of\r\nenvironmental factors on morphology in the future. Using BLADE to overexpress MinD10, I\r\ndemonstrate that minicell formation can be triggered at a specific time point, which offers the\r\npossibility to obtain minicells of similar metabolic activity, that bears potential benefits for the use of\r\nminicells as delivery vehicles. To showcase the advantage of light as an external trigger in medium7\r\nand high-throughput assays, I built a library of 117 constructs to characterize 39 E. coli genes with\r\nunknown or poorly defined function in terms of intracellular localization and effect on cell growth\r\nand morphology of their overexpression. I identified several proteins that, when overexpressed, affect\r\ncell growth, both positively and negatively; I also found two proteins whose overexpression leads to\r\ncell elongation and another one that exhibits a toxic effect. Lastly, through fusion to a fluorescent\r\nreporter, I determined their localization.\r\nIn conclusion, I believe that BLADE is a robust and effective optogenetic tool for the study of\r\nbacterial gene regulatory networks and gene function. I expect that its plug-and-play functionality,\r\ntogether with its tight induction control and its reliable performances, will allow its adoption in\r\nmicrobiology, synthetic biology and biotechnology."^^ . "2021" . . . . . . . "Edoardo"^^ . "Romano"^^ . "Edoardo Romano"^^ . . . . . . "Engineering AraC to make it responsive to light instead of arabinose (PDF)"^^ . . . "PhD Thesis Edoardo Romano.pdf"^^ . . . "Engineering AraC to make it responsive to light instead of arabinose (Other)"^^ . . . . . . "indexcodes.txt"^^ . . . "Engineering AraC to make it responsive to light instead of arabinose (Other)"^^ . . . . . . "lightbox.jpg"^^ . . . "Engineering AraC to make it responsive to light instead of arabinose (Other)"^^ . . . . . . "preview.jpg"^^ . . . "Engineering AraC to make it responsive to light instead of arabinose (Other)"^^ . . . . . . "medium.jpg"^^ . . . "Engineering AraC to make it responsive to light instead of arabinose (Other)"^^ . . . . . . "small.jpg"^^ . . "HTML Summary of #30010 \n\nEngineering AraC to make it responsive to light instead of arabinose\n\n" . "text/html" . . . "000 Allgemeines, Wissenschaft, Informatik"@de . "000 Generalities, Science"@en . . . "570 Biowissenschaften, Biologie"@de . "570 Life sciences"@en . .