Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Room: Exhibit Hall
Sophia Liberto - University of Maryland, Baltimore County
Co-Author(s): Robin Bridgman, University of Maryland, Baltimore County, MD; Stephen Miller, University of Maryland, Baltimore County, MD
Understanding interactions between the algal genome and environmental conditions is fundamental to the goal of generating strains that can become economically beneficial and environmentally sustainable players in the future of energy production. Algae produce the lipid triacylglycerol (TAG) as a stress response to environmental conditions; the TAG produced is of interest due to its conversion into biodiesel. The purpose of this study is to investigate the mechanism behind TAG compensation observed in knockout mutants of TAG synthesis genes by crossing a upf3 mutation into previously generated mutants of Chlamydomonas reinhardtii (Lee et al., 2021). The UPF3 protein is involved in the nonsense-mediated decay mechanism (NMD) and is proposed to influence genetic compensation. Genetic compensation is when a knockout mutation induces the transcription of a different gene that serves the same function, therefore maintaining the needs of the organism. The two genes of interest are phospholipid: diglycerol acyltransferase (PDAT) and the vacuolar transport chaperone (VTC1). PDAT is involved in the acyl-CoA independent pathway of TAG synthesis in C.reinhardtii, and the VTC complex helps sequester polyphosphate chains in vacuole-like structures to maintain homeostasis and cellular energy. For desired progeny, mutant strains of pdat and ∆vtc1/pdat were crossed to a upf3 mutant. The controls used to visually compare mutant alleles observed in crossed vs. non-crossed strains through PCR consist of CC-4533, upf3, pdat1-1, and ∆vtc1/pdat. So far, we have determined optimal PCR conditions to positively identify mutations and have promising results for a potential pdat/upf3 cross. Once pdat/upf3 and ∆vtc1/pdat/upf3 mutants are confirmed through PCR analysis, strains will be grown in N and P deprived media to induce TAG gene expression and TAG production. TAG gene expression will be measured by RT qPCR and TAG content by thin-layer chromatography (TLC). The results will be used to assess the involvement of NMD in the compensation of TAG seen in the ∆vtc1/pdat mutants. The pdat/upf3 results will aid in the further investigations of PDAT upregulation seen in additional TAG gene knockout mutants. Exploration of this genomic response will give insight into the pathway of genetic compensation seen in TAG gene mutants and potentially further the development of cost-effective algal strains for biofuel production.
Funder Acknowledgement(s): This study was supported by funding from the REM supplement to NSF award 1332344.
Faculty Advisor: Stephen Miller, email@example.com
Role: I worked on confirming mutations present in control strains with PCR analysis, optimizing PCR conditions to identify either wild-type or mutant genes, performing crosses of pdat and vtc/pdat with upf3, and PCR analysis of cross progeny.