My lab develops and uses tools based on unnatural amino acids to probe neuronal circuits in C. elegans worms.
Our aim is to employ this novel class of tools towards answering one of the biggest open questions in science: how do a nervous system’s components work together to acquire sensory inputs from the environment and then compute this information to produce behavioural outputs?
The genetic code consists of 64 triplet codons of which 61 code for the canonical 20 amino acids and the remaining three signal termination of protein synthesis.
Even though this code and the machinery to translate it into proteins is extremely conserved across all life forms, methods have emerged in recent years to artificially expand the genetic code allowing us to incorporate chemically synthesized “unnatural” designer amino acids into proteins in living cells.
In its most simple form, genetic code expansion to incorporate unnatural amino acids (UAA) site specifically into proteins requires an orthogonal aminoacyl tRNA synthetase/tRNA pair to be introduced into the host organism.
The orthogonal aminoacyl-tRNA synthetase must specifically recognize an unnatural amino acid and use this amino acid to specifically charge its cognate orthogonal tRNA, which is itself not a substrate for endogenous synthetases.
The charged tRNA then decodes an amber stop codon introduced into a gene of interest at a specific site. UAA open the door to dissecting biological processes inside living cells with the precision, temporal resolution and directness typically associated with in vitro biochemical experiments.
Examples of UAA currently available for use include photo-caged amino acids, which allow the design of light-activatable proteins and have, for example, been used to construct light activatable kinases; or amino acids carrying bioorthogonal chemical handles, which allow site specific labeling of proteins but have recently even been used to label proteomes for tissue specific proteomics studies.
The use of unnatural amino acids (UAA) had been limited to single celled systems until very recently, when we were able to extend the technique to multi-cellular organisms, making C. elegans the first ever animal with an expanded genetic code.
C. elegans is a small nematode worm and a favourite model organism in neurobiology. With an extraordinarily compact nervous system, made up of only 302 neurons, it is able to respond to a broad range of environmental cues with a surprisingly complex array of behaviours.
The complete anatomical wiring diagram of the worm’s nervous system was established more than 20 years ago, however knowledge of the physical connectome turned out to be insufficient to understand the worm’s behaviour on its own, and progress on functional data, as is the case in other neurobiological models, has been slow due to the lack of specific tools.
We use photo-caged amino acids to create light activatable protein variants, allowing us to directly control targeted proteins and neurons in vivo and even in freely moving animals.
We then study the contribution of individual components of the nervous system to behaviour by observing the animals’ response to environmental stimuli before and after photo-activation.
Elliott TS, Townsley FM, Bianco A, Ernst RJ, Sachdeva A, Elsässer SJ, Davis L, Lang K, Pisa R, Greiss S, Lilley KS, & Chin JW (2014). Proteome labeling and protein identification in specific tissues and at specific developmental stages. Nature Biotechnology 32, 465-472
Radman I, Greiss S§, & Chin JW§ (2013). Efficient and Rapid C. elegans Transgenesis by Bombardment and Hygromycin B Selection. PLoS ONE, 8(10), e76019.
Bianco A*, Townsley FM*, Greiss S*, Lang K & Chin JW (2012). Expanding the genetic code of Drosophila melanogaster. Nature Chemical Biology, 8(9), 748-750. (Featured on New Scientist)
Greiss S & Chin JW (2011). Expanding the Genetic Code of an Animal. Journal of the American Chemical Society, 133(36), 14196-14199. (Featured on the BBC, BBC World Service Radio, Chemistry World, ChemBiochem. 2012)
Pourkarimi E, Greiss S, & Gartner A (2011). Evidence that CED-9/Bcl2 and CED-4/Apaf-1 localization is not consistent with the current model for C. elegans apoptosis induction. Cell Death and Differentiation, Mar; 19(3):406-15.
Ito S, Greiss S, Gartner A, Derry WB (2010). Cell-nonautonomous regulation of C. elegans germ cell death by kri-1. Current biology, Feb 23; 20(4):333-8
Greiss S, & Gartner A (2009). Sirtuin/Sir2 phylogeny, evolutionary considerations and structural conservation. Molecules and Cells, 28(5), 407-415.
Greiss S, Hall J, Ahmed S, Gartner A (2008). C. elegans SIR-2.1 translocation is linked to a proapoptotic pathway parallel to cep-1/p53 during DNA damage-induced apoptosis. Genes & Development, Oct 15; 22(20):2831-42.
Greiss S*, Schumacher B*, Grandien K, Rothblatt J, Gartner A (2008). Transcriptional profiling in C. elegans suggests DNA damage dependent apoptosis as an ancient function of the p53 family. BMC Genomics, Jul 15; 9:334.
*equal contribution § co-corresponding author
Chin, J., Deiters, A., Greiss, S., Hancock, S. M., Uprety, R. (2013). Methods for Incorporating Unnatural Amino Acids in Eukaryotic Cells. Patent Application WO2012038706 A1