Generation and Characterization of Transgenic Chlamydomonas reinhardtii Overexpressing the Novel Gene Cia7

Abstract

The constant increase in environmental pollution, in particular heavy metal pollution is alarming. Cia7, the gene understudy has conserved cysteine residues. These residues could be involved in metal binding; and the CIA7 protein might be associated to metal homeostasis in Chlamydomonas reinhardtii. The successful overexpression of Cia7 in C. reinhardtii could enhance its metal tolerance. Current research in bioremediation and heavy metal pollution is present in the literature. However, there is limited research in this area that involves C. reinhardtii. Additionally, the function of CIA7 is still unknown. The core hypothesis is that CIA7 plays a role in metal tolerance and in metal binding. This research will attempt to address if CIA7 performs a role in C. reinhardtii’s tolerance to metal toxicity. The aim of this research is to characterize the Cia7 gene and to assess its potential role in metal homeostasis. The objectives of the research are (1) to overexpress CIA7 in C. reinhardtii, and (2) to compare cell growth, chlorophyll content and bioaccumulation of heavy metals in cc5013 mutant, cc4425 wild‐type and the selected transformants under the presence or absence of lead. The gene was overexpressed in wild‐type (WT) and mutant (MT) C. reinhardtii cells.WT and MT cells were subjected to lead (Pb) assays in order to find the optimum sub‐lethal concentration. Once the optimum sub‐lethal concentration was found, sub‐lethal assays were performed. The goal of the sub‐lethal assays was to observe the growth of transformants and to observe any difference in phenotype among cells with the overexpressed Cia7 gene, the WT and the MT when exposed to metal stress. To establish whether exposure to metal would affect chlorophyll biosynthesis, a chlorophyll content analysis was conducted. To measure lead bioaccumulation among the strains, inductively coupled plasma‐optical emission spectrometry (ICP‐OES) analysis was conducted. Lastly, cell density was recorded to measure and compare the growth of the different C. reinhardtii strains. Samples for all of these protocols were collected at different time intervals. The sub‐lethal concentration of lead, whereby a difference in growth was observed was at 1.5 mM Pb. Interestingly, there were colonies from the generated transformants that were found to be more lead tolerant at 1.5 mM compared to the wild‐type and the mutant strains. These colonies, the wild‐type and the mutant strains were subjected to a chlorophyll content analysis and bioaccumulation analysis to determine if there is any difference among them. Results from these experiments will be reported. The findings of this research could provide a basis to (1) use Cia7 as a biomarker for metal contamination, or to (2) make the CIA7 protein be overexpressed in C. reinhardtii or in another organism for use in phytoremediation.Support or Funding InformationA special thanks to my research mentor, Dr. R Ynalvez. Thank you to TAMIU Graduate Financial Assistance for my assistantship and to the Office of Research and Sponsored Projects for my summer 2019 fellowship. Thanks to Dr. M Ynalvez for the statistical analysis. Thank you to fellow lab mates for the support in this research.