1. Mentor: Kirsten Crossgrove, PhD
Using Bioluminescent and Fluorescent Organisms as Environmental Sensors
Amanda Danno
Mentor: Kirsten Crossgrove, PhD
University of Wisconsin – Whitewater, Department of Biological Sciences
Introduction
Reporter genes are highly useful in the biotechnology field. Bioluminescence and fluorescence are both examples of the output of reporter gene systems. Bioluminescence occurs when enzymes catalyze light releasing reactions, whereas fluorescent proteins release light of one wavelength when excited by light of another wavelength. We are developing a kit that teaches about reporter genes. In this kit, the model organism Caenorhabditis elegans, a free-living nematode, will be used to detect the presence of heavy metal or other contaminants in water using bioluminescence and/or fluorescence. Heavy metals pose threats to human health and are commonly found in the environment. C. elegans can serve as a model for multicellular organisms due to its sensitivity to heavy metals and conserved stress response signaling pathways. We are currently conducting ‘proof of principle’ experiments to see if either a bioluminescent or fluorescent reporter gene system is amenable to classroom experiments.
Hypothesis
We predict C. elegans can be used as a biosensor organism to teach about reporter gene systems in lab activities to high school and introductory college students.
Research Question
Will bioluminescence and fluorescence be able to produce a quantitative and visual qualitative representation of reporter genes in the C. elegans system?
Using C. elegans in the classroom:
Sources and effects of heavy metals:
Current experimental design:
Bioluminescence
We have obtained transgenic C. elegans (strains PE254 and PE255) that express luciferase at high levels in all tissues (Lagido et al., 2001). .
In the presence of heavy metals, the amount of light released is reduced due to mitochondrial stress, which lowers ATP levels (Lagido et al., 2001). Increasing concentrations of copper sulfate (CuSO4) reduced the amount of light, and increased lethality in transgenic C.elegans.
Summary and conclusions:
We can use bioluminescence to measure high concentration of heavy metals and see effects, but we have not been able to capture the bioluminescence using a smart phone camera. The bioluminescence system produces consistent results, although it is not reliable to test small amounts of heavy metals. The system would not suffice for a classroom setting because it is not sensitive enough to measure realistic amounts of heavy metals.
Fluorescence
We obtained a transgenic strain of C. elegans (ST66) which contains a hsp16.2 (heat shock) promoter controlling expression of enhanced green fluorescent protein (eGFP).
eGFP should be produced when the worms are stressed (Anbalagan et al., 2012). Exposure to CuSO4 or heat sometimes increases eGFP production.
Summary and conclusions:
This system has proved to be insufficient for a classroom setting because the amount of eGFP observed was not consistent, and the eGFP couldn’t be easily observed without the use of a fluorescent microscope. A. B.
Figure 7: The structure of eGFP is shown. A single amino acid substitution in the original GFP gene results in a protein with brighter fluorescence.
Figure 4: The luciferase gene drives expression of the luciferase enzyme in all cells (A), but the luciferin substrate and ATP are needed to allow luciferase to produce light (B). Images adapted from: C. B. A. B. C.
Figure 8: Fluorescence can be visualized using a fluorescent microscope (A), measured using the GlowMax luminometer (B) or viewed by excitation using a handheld UV light (C). A. *
Figure 1: Caenorhabditis elegans have a three day life cycle at room temperature (A), can be maintained on agar plates containing bacteria as their food (B) and are easily visible under the light microscope (C).
Figure 9: This shows C. elegans with the GFP activated under the fluorescence microscope. A. B.
Figure 5: Dilution series of CuSO4 in the luminescence strain (PE255). Bioluminescence was measured in relative light units taken from a GlowMax luminometer. Worms were incubated with shaking for 24hours and 30 minutes before readings were taken. * p < 0.05
Figure 2: Heavy metals affect the oxidative processes in cells and cause apoptosis (cell death) (A) Image adapted from Jaishankar, et. al (2014). There are many environmental sources of heavy metals (B).
Acknowledgments
Austin Chriske, Kristin Hausen and Johnna Dykstra for help in the lab
UW-Whitewater Undergraduate Research Program for funding
UW-Whitewater Department of Biological Sciences
Caenorhabditis Genetics Center (CGC), University of Minnesota, Saint Paul for transgenic worms.
References
Lagido, C., Petitt, J., Porter, A. R., Paton, G., & Glover, L. (2001). Development and application of bioluminescent Caenorhabditis elegans as multicellular eukaryotic biosensors. FEBS Letters, 493: doi: /
Anbalagan, C., Lafayette, I., Antoniou-Kourounioti, M., Gutierrez, C., Martin, J. R., Chowdhuri, D. K., & Pomerai, D. I. (2012). Transgenic nematodes as biosensors for metal stress in soil core samples. Ecotoxicology, 21: doi: /s
Jaishankar, M., Tseten, T., Anbalagan, N., Mathew, B. B., & Beeregowda, K. N. (2014). Toxicity, mechanism and health effects of some heavy metals.Interdisciplinary Toxicology, 7(2). doi: /intox
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Image:
Image adapted from
Images: 5 1 2 3 4
Figure 6: This is an image from a CCD camera using ImageQuant software that visually shows the bioluminescence. Well 1 contains no CuSO4, well 2 contains M, well 3 contains M, well 4 contains M and well 5 contains 0.25M.
Figure 3: The flow chart explains our current protocol. In developing the kit we hope to simplify the steps and equipment needed to make it feasible for high school classrooms.