[
International Worm Meeting,
2019]
The purpose of this study is to create a Caenorhabditis elegans (C. elegans) mutant library by using CRISPR/Cas9 to knock out UGT genes. This library will be comprised of the existing UGT mutants in order to provide us with the needed information to peruse other non-explored UGT genes to knock out in the future. In C. elegans, UGT genes regulate the glycosylation of environmental toxins allowing for survival of the nematode[1]. CRISPR/Cas9 is a powerful gene-editing system allowing for a Cas9 endonuclease to induce a double strand break in the DNA, rendering non-homologous end joining between the broken DNA[2]. As a result, that particular gene in the DNA is knocked out and a mutant is created. As part of the Vertically Integrated Projects (VIP) undergraduate research team at UGA, we have developed a workflow that will allow us to create this mutant library[3]. Upon completion, this library will allow us to test the effects of different xenobiotics and natural compounds on UGT knockout mutants which will allow us to better understand the role of these genes and their associated proteins in the glycosylation and drug resistance pathways of C. elegans; this provides us with a model which can be later be tested in parasitic nematodes. Additionally, the CRISPR/Cas9 protocols established for UGT knockouts will allow future undergraduate students to partake in CRISPR/Cas9 genetic research through the VIP program in the Edison Lab to continue producing UGT mutants for metabolomics analysis.
[
Evolutionary Biology of Caenorhabditis and Other Nematodes,
2010]
Virtually all organisms live in a heterogeneous environment. It is commonly assumed that phenotypic plasticity is associated with environments variable in space and/or time, and that such a 'Jack of all trades' genotype would do better than a fixed genotype. On the other hand, a genotype with phenotypic plasticity is expected do worse in a constant environment than a fixed would. The cost of maintaining phenotypic plasticity is commonly associated with the maintenance and production of genetic and cellular machinery to detect and produce the best phenotype for the environment. Although the idea of the cost of maintaining phenotypic responses in a constant environment is widely recognised, it has not been demonstrated experimentally. We addressed this question by using long-term selection experiments on a gonochoristic nematode species (Caenorhabditis remanei). Initially, replicates of worms were maintained for 50 generations under two temperature regimes: constant temperature (mean 15C) and fluctuating environment with the same mean but temperature fluctuating between 5 and 25C every 12 hours. The objective of this experiment was to select for individuals with different levels of plasticity. Plasticity levels were measured by comparing the ability of a line to maintain high fitness across a temperature gradient. After 50 generations in each environment, populations were transposed between these environments. The objective was to compare differences in fitness of individuals from the two regimes before and after the selection experiment. Comparisons of fitness across the environments will enable to determine if selection for plasticity is costly in a constant environment, and if specialisation to a constant environment carries a cost when transferred to a fluctuating environment. The results of the first experiment showed changes in fitness across temperatures; worms from a fluctuating environment showed wider thermal breath compared to worms selected for a constant environment. This suggests that phenotypic plasticity was favoured in a fluctuating environment and it had some genetic basis. Comparisons of fitness before and after the selection experiment showed that increased phenotypic plasticity potentially incurred a fitness cost. Worms cultured in a fluctuating environment for 50 generations showed reduced fitness when cultured in a constant environment compared to worms in a constant environment at the beginning of the experiment. However, worms cultured in the constant regime and moved to the fluctuating environment showed no differences with worms in a fluctuating environment at the beginning of the experiment. These results suggest that, for this system, there is a potential cost of adapting via phenotypic plasticity but there is not a cost for becoming a fixed genotype.