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Establishing genetic code expansion as a tool to study neuronal circuit function in an animal (UAAWORM)
Start date: Apr 1, 2016, End date: Mar 31, 2021 PROJECT  FINISHED 

We work at the intersection of synthetic multicellular biology and organismal neurobiology. We pioneered technologies that allow the site-specific introduction of chemically synthesized unnatural amino acids (UAA) into a chosen protein within the context of a multicellular organism - the nematode worm C. elegans. The foundational technology for this advance is genetic code expansion. In its most simple form, genetic code expansion to incorporate UAA into proteins requires an orthogonal aminoacyl-tRNA synthetase/tRNACUA pair to be introduced into the host organism. The orthogonal aminoacyl-tRNA synthetase must specifically recognize an UAA and use this amino acid to specifically aminoacylate its cognate orthogonal tRNACUA, which is itself not a substrate for endogenous synthetases. The aminoacylated tRNA then decodes an amber stop codon introduced into a gene of interest at a specific site.We aim to engineer photo-activateable site-specific DNA recombinases by incorporating a photo-caged amino acid into the active site of the recombinase. We can then use a microscope mounted laser to uncage and activate it in single C. elegans neurons. This will allow us to selectively control expression of i) proteins to control neuronal function (e.g. optogenetic channels, Tetanus toxin, etc.) and ii) endogenous neuronal proteins. We will use this ability to: i) control within an intact, freely moving animal the activity of any desired single neuron or group of neurons, and ii) precisely shape and remodel the chemical and electrical synaptic connectivity between neurons. We will then apply this technology to investigate how neuronal circuits generate behaviour. In addition and complementary to the development and application of neurobiological tools we will further expand the coding capacity of the worm’s protein synthesis machinery by i) introducing additional UAAs to C. elegans, and ii) going beyond triplet codons to engineer animals capable of decoding quadruplet codons.
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