Green Fluorescent Protein

Overview

Objectives/Goals To determine if inorganic mercury is able to penetrate the cell membrane of mammalian neurons. Methods/Materials Materials: The Mutant GFP and mercury (HgCl2)The fluorometer, fluorescence microscope, sonicator, centrifuges, UV spectrophotometer, pH meter, rat brain neurons. We will use a fluorescence microscope to visualize the light emitted from the rat neurons. The fluorescence microscope will be used at UCSB. Mercury (HgCl2), Buffer, Salt, Culture media, Virus. Methods: A. Treatment of Purified GFP with Mercury Measure Fluorescence Emission (Using a fluorometer) # Ignite the xenon lamp with 40,000 volts # Load GFP sample into cuvette # Load cuvette into fluorometer # Determine the excitation wavelength maximum by detecting fluorescence emission at 512nm and then scanning the excitation wavelengths from between 350nm to 500nm (Fig. 7) # Set excitation wavelength to 466 nm for all experiments. # Add mercury to GFP in different amounts # Record the fluorescence emission at 513 nM (Fig. 8) Treatment of Neurons with Mercury Expression of GFP in Mammalian Neurons # GFP gene is cloned into a virus for infection of neurons # Neurons are taken from the brains of rat fetuses and kept alive in cell culture # Then neurons are infected with the GFP-containing virus # The GFP protein is expressed in the neurons # Mercury is added to the neurons and GFP fluorescence was then examined under a fluorescence microscope (Figs. 10 & 11) # Pictures were taken and analyzed Results We found that the inorganic mercury was not able to penetrate in the 5 minute time limit we gave it but only after 9 hours did the mercury get into the neurons. Conclusions/Discussion the mercury was not able to penetrate the cell membrane

Awards (1)

• Honorable Mention

Competition history

• CSEF 2009 Toxicology (Junior Division Only) · Entry J2313

Resources

• CSEF project summary ↗

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