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Lighting up Biology: Expression of the Green Fluorescent Protein

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Lighting up Biology: Expression of the Green Fluorescent Protein Lighting Up Biology: Expression of the Green Fluorescent Protein By Martin Chalfie and Ghia Euskirchen A Key Experiment produced by The Explorer’s Guide to Biology 2 Th e Explorer’s Guide to Biology https://explorebiology.org/ Lighting up Biology Expression of the Green Fluorescence Protein Martin Chalfi e and Ghia Euskirchen Martin (Marty) Chalfi e Martin (Marty) Chalfie holds the title of University Professor at Columbia University in New York City. He shared the 2008 Nobel Prize in Chemistry, along with Osamu Shimomura and Roger Y. Tsien, “for the discovery and development of the green fl uorescent protein, GFP.” He received his Ph.D. in Physiology from Harvard University in 1977. ghia Euskirchen Ghia Euskirchen received a Ph.D. in molecular biology from Columbia University. Her career has taken her to Yale University as a postdoc, then to Stanford University where she was director of a DNA sequencing center, and she currently resides in Washington, DC. Published on September, 2019 3 Lighting up Biology: Expression of the Green Fluorescence Protein What’s the Big Deal? Certain jellyfish emit a beautiful green light. Although why jellyfish would want to glow in the dark is still a mystery, the green fluorescent protein (GFP) from these animals has proven to be a gift from nature that has transformed cell biology. Three scientists were recognized with a Nobel Prize for their work on GFP, although others contributed in important ways, as you will see from this story. Why is GFP a big deal? With the combination of GFP and microscopes equipped with very sensitive cameras, scientists began making incredible movies of the dynamic processes that underlie life at that microscopic level. Perhaps you have seen “Planet Earth” and the amazing cinematography of animals in these videos. Now, equally amazing movies are being made of the inner workings of living cells, allowing us to see when and where genes are expressed and other dynamic processes that enable life. GFP has given us the tools to make “Planet Cell.” GFP also unleashed the amazing creativity of scientists. GFP has been engineered into “endless forms most beautiful and most wonderful” (to quote Darwin). There are now fluorescent proteins of all colors, variants that can be switched on and off, and modified versions that sense and report upon condi- tions inside of cells. And it all started with a deceptively simple experiment. The story of GFP also contains a lesson for young scientists—don’t be dissuaded by other people who might say “no, that experiment won’t work.” We will let Marty Chalfie and Ghia Euskirchen take you on a tour of what they did, giving you a front row seat to the birth of the GFP revolution. Learning Overview Big Concepts The “green fluorescent protein” (GFP) has revolutionized cell biology because it provides a fluores- cent beacon that enables scientists to follow individual molecules in a living cells or the behavior of whole cells in living organisms. This key experiment (first done by a new graduate student) also provides a window into the process of gene cloning, how GFP is being used in biology, and into the serendipitous nature of science itself. Terms and Concepts Used Amino acids, antibody, bioluminescence, Central Dogma, DNA, enzyme, fluorescence, gene, mRNA, mutant, neuron, plasma membrane, protein, protein phosphorylation Terms and Concepts Explained C. elegans, DNA cloning, E. coli, fluorescence, immunofluorescence, mitotic spindle, polymerase chain reaction, reporter, transformation, restriction enzyme 4 Lighting up Biology: Expression of the Green Fluorescence Protein Key Experiment Introduction Certain jellyfish (including Aequorea victoria) possess a protein that can absorb blue light and emit green light. This protein is called the green fluorescent protein or GFP. What Events Preceded the Experiment? Before GFP, scientists used a technique called “immunofluorescence” to reveal the location of a protein within a cell. However, this method involved chemical fixation, which killed the sample a and precluded observation of cellular dynamics. If GFP could be expressed in living cells other than the jellyfish, then live cell observations could be made. Setting up the Experiment Martin Chalfie, his student Ghia Euskirchen, and collaborator Douglas Prasher undertook an experiment to express the cloned jellyfish GFP DNA in bacteria and see if the living bacteria would glow green. Many people thought that this experiment would not work. GFP undergoes a chemical reac- tion that produces a ring within its original linear backbone, which is critical for its fluorescence. Several researchers at the time speculated that one or more jellyfish enzymes might be needed for this reaction. If this speculation were true, bacteria lacking these hypothetical enzymes could not fluoresce green. Doing the Key Experiment The scientists used polymerase chain reaction and cDNA cloning to introduce the jellyfish GFP gene on a plasmid into bacteria. Bacteria that successfully took up the plasmid were selected using an antibiotic resistance marker. The scientists induced the expression of the GFP gene. A control experiment was performed in which bacteria were transformed with a plasmid that did not contain the GFP gene. They looked to see if the bacteria with the GFP gene became fluorescent whereas the control bacteria did not. The experiment worked on the first try. The bacteria with the gene, but not the control, became fluorescent. No additional enzymes from the jellyfish were needed. GFP then was used as a “transcriptional reporter” to analyze what genes are turned on in partic- ular cells. The GFP gene also was joined onto other genes to create “fusion proteins”, where the GFP fluorescence could be used to track the whereabouts of proteins in cells. 5 Lighting up Biology: Expression of the Green Fluorescence Protein What Happened Next? New fluorescent proteins with different colors were discovered or made. Numerous uses of GFP evolved in biotechnology and cell biology. Because of the importance of GFP as a tool, three sci- entists were awarded the Nobel Prizes for discoveries related to GFP. Closing Thoughts The GFP story reveals how important new technologies can emerge from studying unusual organ- isms (jellyfish), why persistence pays off, and how serendipity takes one down unexpected paths of discovery. Guided Paper M. Chalfie, Y. Tu, G. Euskirchen, W.W. Ward, and D.C. Prasher. Green fluorescent protein as a marker for gene expression. Science 1994;263:802–805. 6 Lighting up Biology: Expression of the Green Fluorescence Protein Introduction I am famous in my department for sleeping through scientific talks, but on April 25, 1989, I heard one that kept me wide awake. I am a faculty member at Columbia University, and for many years, those of us interested in neuroscience met every Tuesday at noon to hear a scientific seminar. That Tuesday, the speaker, Paul Brehm, grabbed my attention. He began by describing the work of Osamu Shimomura on bioluminescence, the ability of different organisms to generate light. I am famous in my department for sleeping through scientific talks I had known of one of Shimomura’s discoveries—the isolation of the bioluminescent protein aequorin from the jellyfish Aequorea victoria. Aequorin produces a bright blue light in the pres- ence of calcium. What I hadn’t known until Paul’s seminar was that Shimomura had discovered a second protein, a protein that he initially called the Green Protein and that we now call the Green Fluorescent Protein or GFP. GFP was going to change my life. Osamu Shimomura’s discovery of these two proteins are themselves a study in overcoming scien- tific frustration. He started out with a fascinating problem: how do different organisms generate light? Many organisms (e.g., fireflies, glow worms, bacteria, fungi, fish) generate light, but they do so in many different ways (see also, the story of bioluminescent bacteria in the Narrative on Quorum Sensing by Bassler). Shimomura wanted to find the molecules that allowed the jellyfish to produce a beautiful and intense green light. However, isolating the light-producing protein from the jellyfish proved elusive. One night, after another frustrating day of failure, he decided to clean up and go home for dinner. He threw all of his seemingly worthless protein samples into a sink that also had an overflow from seawater tanks. Shimomura turned off the lights and was about to walk out the door, when he looked back at the sink. It was glowing! Something from his protein samples was generating light. As you can imagine, literally seeing the light made him stop and think about what was happening. After some thought, he realized that it must have been the seawater that mixed in with his protein and caused light to be produced. Seawater contains calcium, something he had never tried adding in his experiments. Perhaps calcium was needed to trigger a jellyfish protein to make light? A few days later he showed that his guess was correct; addition of calcium to his preparation produced a flash of light. He then used this ability to stimulate light production to guide him in the purifica- tion of the aequorin protein. It was glowing! Something was generating light. Unfortunately, Shimomura’s discovery of aequorin left him with another problem: the light emit- ted by aequorin was blue instead of the green glow emitted by the jellyfish. Some other factor (another protein perhaps?) must have converted the blue light to green. When he tested other jellyfish protein samples, he indeed found another protein that could absorb blue light and re-emit it as green light. That protein we now call GFP. 7 Lighting up Biology: Expression of the Green Fluorescence Protein VidEo 1. The Expression of the Green Fluorescence Protein. Martin Chalfie and Ghia Euskirchen describe their experiment. What events preceded the experiment? When I heard about GFP in that seminar in 1989, I was so excited that I spent the rest of the semi- nar time thinking about the experiments I wanted to do and was not paying attention to the speaker.
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