Biotechnology: Green Fluorescent Protein Grade levels: High School

How did a jellyfish end up producing one of the most important scientific discoveries of all time?

Introduction Video http://www.scienceinseconds.com/Brain-and-Behaviour/GFP What does GFP stand for? Green Fluorescent Protein

Bioluminescent Organisms Occur In Nature Bioluminescence: when living organisms (bio) give off light GFP was first discovered in the jellyfish  Aequorea victoria Can you name other animals that naturally glow in the dark? Bioluminescence is found in many other organisms: microbes, algae, fireflies, glow worms, etc. https://www.nobelprize.org/nobel_prizes/chemistry/laureates/2008/illpres.html

Scientists won the Nobel prize in 2008 for their discovery of GFP Images are photos of the 3 scientists, and the protein structure of GFP. GFP protein glows green when ultraviolet light is shone on it UV light is absorbed; green light is reflected back to our eyes

Scientists can create transgenic, glowing organisms by “tagging” genes with GFP DNA of gene x (+ GFP gene) is transcribed into RNA, and then translated into a GFP fusion protein! Normal protein product of the gene, but with GFP attached. https://www.nobelprize.org/nobel_prizes/chemistry/laureates/2008/illpres.html Unlike other labels, GFP can be done to living cells! It will not alter normal cell activity. Why is this beneficial for scientific study?

Scientists have found fluorescence in every color of the rainbow! As students why multiple colors would be advantageous? (Study more than one label at once, ex different parts of the cell, multiple cell types, different proteins… etc).

How can science use this to study biology?: Whole Organism Development Label cells during development Study cell cycle, growth, maturation https://www.youtube.com/watch?v=FChS4KU5jDM All of the nuclei have been labeled, so we can watch the cells during fruit fly embryogenesis Do the cells stay in one place, or do they move? Do they change size or shape? Are there always the same number of cells, or does it change? Developmental biology: how cells in an early life stage correspond to cells/tissues/organs in an adult

How can science use this to study biology?: Study Specific Cell Types In this image, one stem cell (*) has been labeled with GFP, and the other has not. As the cells divide, the label is retained. In this tissue, from left to right we can visualize a timeline of stem cell differentiation. All of the GFP labeled differentiated cells originated from that original stem cell. * * This image is from my own work visualizing Drosophila melanogaster (fruit fly) ovaries. We study how germline stem cells (indicated here with a *) differentiate to form mature oocytes (egg cells). More information can be found here https://blog.ecu.edu/sites/ablese/. For an in depth lesson plan on stem cells, I direct you to my lab mate Taylor Hinnant, whose lesson plan “Learning about cell differentiation using blood cells” is also available through the SciREN portal. Why do scientists study stem cells?

Fluorescence can be used to visualize mitosis! Can you identify different parts of the cell? Cytoplasm? Chromosomes? Watch the fluorescently labeled cells undergo mitosis! https://www.youtube.com /watch?v=FPtFXNmhI_g The line in the video, called a “scalebar”, acts like a ruler to show how tiny the cells are. 25 microns= 0.025 milimiters=0.000025 meters! The video link is quite short- you may need to play it more than once. Chromosomes are labeled in yellowish green, spindle is in blue. Ask students to count how many cells are visible at start and at end. Have them guess which cells are in what stages (interphase, metaphase, anaphase, cytokinesis). If not previously covered, more cell cycle information can be found here http://biology.tutorvista.com/cell/mitosis.html. https://micro.magnet.fsu.edu/cells/fluorescencemitosis/

Identify Cell Cycle phases Identify Cell Cycle phases! Here the DNA (chromosomes) have been labeled with GFP Have the students identify some different phases of the cell cycle. Interphase is the most common, but you can also easily point out metaphase (where chromosomes line up in the middle of the cell(arrow, animated on click). Original paper is here http://www.cell.com/fulltext/S0960-9822(98)70156-3

Even tiny pieces of cells can glow in the dark! https://www.youtube.com/watch?v=YyFQ51bayws This video shows red mitochondria (provides energy for the cell) moving along a cell’s green cytoskeleton (microtubules that give a cell its shape). Can you name some other cell organelles? What is their function? How can visualizing organelles help us to understand how they work? Images from https://www.thermofisher.com/order/catalog/product/C10505?ICID=search-product In images and video mitochondria are shown in red. The large “hole” in the middle of some images are where the nuclei (unlabeled) are. These are mammalian cells.

Now It’s Your Turn Pretend you are a scientist: a biotechnologist! You are a GFP guru. You can study any organism, for any reason What is your hypothesis? Do you want to learn more about a disease, and how it affects the body? Do you want to learn about a certain type of tissue or cell? Do you want to learn about cell organelle(s)? What do you want to make glow in the dark? What would you expect to see? Draw or write about your plans Do some googling! Maybe other scientists are working on your questions right now! If desired, refer to the attached worksheets. This can be done in class, or as a homework assignment. Depending on the size of the class, this could be an individual project or groupwork. Sample project idea: Maybe a student is interested in sickle cell anemia. What blood cell type should they focus on? (erythrocytes, red blood cells). Compare and contrast (including ethical considerations) of using human cells vs animal models. Should they label a specific part of the cell (ex cytoskeleton to visualize cell shape, hemoglobin content etc.)?

Punnett Square Template http://www.studenthandouts.com/01-Web-Pages/001-Pages/Punnett-Square-Printable.htm

Punnett Square Key GFP tagged parent (Gg) G g Gg gg g Wild type (no GFP)(gg) See attached worksheet for more information Expected phenotype and genotype ratio 1:1 g gg Gg