Poster abstracts
Poster number 104 submitted by Vy Nguyen
Measuring the Effect of D-Ala vs. L-Ala on E. coli Lacking their Inherent D-Amino Acid Defense Enzyme
Ngoc Tuong Vy Nguyen (Biology Department and Chemistry Department, Earlham College), Dawt Sang (Biology Department and Chemistry Department, Earlham College), Lexie Kuzmishin Nagy, Ph.D. (Biology Department and Chemistry Department, Earlham College)
Abstract:
During translation, the process of cellular protein synthesis, aminoacyl-tRNA synthetases charge tRNAs with the corresponding amino acid [1,2]. Although there are two conformations of amino acids, L- and D-, only L-amino acids are used by cells for protein formation. Mischarged D-aminoacyl-tRNAs can raise D-amino acid toxicity by decreasing the available tRNA to bacteria cells [3]. In E. coli DTD serves as a natural defense against D-amino acid toxicity by removing D-amino acids from mischarged tRNAs [4]. Similarly, rhizobia encode the enzyme ProXp-x, which preliminary data has shown to remove incorrect L- and D-amino acids in vitro, outside a cell, but this protection has not ever been tested in vivo, inside a cell. The goal of our research is to determine if the ProXp-x protects against D-amino acid toxicity in the well-studied E. coli cell. We hypothesize that ProXp-x that is transgenically expressed in E. coli will be able to protect against D-amino acid toxicity. Currently, we are working on establishing phenotypic differences between two strains of Keio Collection Knockout E. coli (parent strain BW25113), one with dtd deleted and one wild type strain [5,6]. We are comparing the growth and viability of these strains in the presence of no amino acids and different concentrations of L and D-amino acids using three different methods: growth curve assays, spotting assays, and colony forming unit (CFU) assays. These results will aid us in determining how different conditions of amino acid concentrations can affect growth since we hypothesize ProXp-x functions similarly to DTD.
References:
References
1. Bacusmo, J. M., Kuzmishin, A. B., et. al (2018). RNA Biology, 15(4–5), 576–585. https://doi.org/10.1080/15476286.2017.1353846
2. Ibba, M., & Söll, D. (2000). In Annual Review of Biochemistry (Vol. 69, Issue Volume 69, 2000, pp. (617–650). Annual Reviews. https://doi.org/10.1146/annurev.biochem.69.1.617
3. Shahjee, H. M., et. al (2002). Journal of Biosciences, 27(5), 515–520. https://doi.org/10.1007/BF02705049
4. Routh, S. B., et. al (2016). PLOS Biology, 14(5), e1002465. https://doi.org/10.1371/journal.pbio.1002465
5. Baba, T., et al. (2006) Mol Syst Biol; 2, 2006.0008. DOI: 10.1038/msb4100050.
6. Sahu, S. R., et. al (2023). Bio-Protocol, 13(21), e4872. https://doi.org/10.21769/BioProtoc.4872
Keywords: D-aminoacyl-tRNA, DTD, trans-editing