Genetically Engineer Yeast to be Fluorescent
AbstractCan you imagine a glowing loaf of bread? You might not be able to make the whole loaf glow, but you can get baker's yeast to fluoresce! The way to do this is to modify the genetic information of the yeast organism. The technology that is used to do this is called genetic engineering. With genetic engineering, you can insert a fluorescent protein gene from a jellyfish into yeast cells, so they start glowing under blue light! Do this project to see for yourself!
To genetically engineer different strains of yeast to make them fluoresce.
Have you ever baked or watched someone bake bread? If so, then you know that yeast is a very important bread ingredient, as it serves as leavening agent and causes the bread to rise. You might be surprised to hear that yeast is actually a living organism! More specifically, yeast is a single-cell eukaryotic microorganism and thus contains complex internal cell structures similar to those of animals and plants. Its scientific name is Saccharomyces cerevisiae (Figure 1). Most eukaryotes are multicellular organisms. Human cells, for example, are eukaryotic cells. This is why yeast is not only important for bread baking, but also plays a major role in biological research. Due to its similarity to human cells, yeast has become a powerful model organism for biochemical and genetic studies. For example, many human genes known to have a role in disease have counterparts in yeast. This allows scientists to test new drugs on yeast cells or to study the effects of certain gene mutations. As a single-celled organism, yeast has the additional benefit that it can be cultured and manipulated using the standard techniques applied to bacteria.
Figure 1. A yeast (Saccharomyces cerevisiae) culture viewed with a scanning electron microscope.
In order to study biochemical processes within Saccharomyces cerevisiae, it is often necessary to alter its original genetic material, such as introducing a certain mutation into a gene. This is achieved by using genetic engineering techniques, like molecular cloning or genome editing. These techniques allow researchers to knock out or delete a particular gene to study its specific function, or to add new genes into a cell to change its properties or to create novel metabolic pathways. Creating a genetically modified organism (GMO) involves multiple techniques and several steps. The techniques applied to yeast are very similar to the techniques applied to bacteria cells. Based on their research goal, genetic engineers usually first identify or choose the gene that they want to modify, insert, or delete. In the next step, all the DNA fragments necessary for the modification of the yeast cell need to be assembled and then introduced into the cell. DNA molecules that are formed by laboratory methods to create new DNA sequences are called recombinant DNA.
The technique for creating and assembling recombinant DNA molecules is called molecular cloning. New or altered DNA sequences can either be synthesized or created by using one of several cloning methods. One of these methods includes cutting and pasting existing DNA pieces together. To cut existing DNA fragments, restriction enzymes are often used, which are able to break apart the DNA at a specific site. Once broken apart, new or altered DNA fragments, or specific genes of interest, can be inserted. The enzyme DNA ligase is then used to fuse the individual DNA fragments of the recombinant DNA together. The next step is to introduce the newly created recombinant DNA into the yeast cell. In molecular cloning, this is usually done with a vector. Vectors are DNA molecules that carry recombinant DNA into the host cell. Once inside the host cell, the introduced DNA can then be replicated and/or expressed. The most commonly used vectors are plasmids (Figure 2). Plasmids are circular DNA molecules that naturally occur in yeast and bacteria. They are usually small, separate from the chromosomal DNA of the cell, and often carry genes that provide a benefit for the cell, such as antibiotic resistance. For genetic engineering, artificial plasmids are created, and the target gene or DNA sequence is incorporated into the plasmid DNA. This way the whole plasmid, including the recombinant DNA, is introduced into the yeast cell.
The first step shows the recombinant plasmid, which has the target gene incorporated in its circular structure. The second step shows the plasmid with an arrow pointing to a schematic drawing of a yeast cell. The yeast cell contains the plasmid with the target gene. The third step shows a schematic drawing of a selective agar plate. Two yeast cells that include the target gene plasmids are shown above the agar plate with arrows that point toward the agar plate. The fifth and last step shows a top view of an agar plate with many circles on it that represent the yeast cells.
Figure 2. Schematic drawing showing some of the steps involved in generating genetically modified yeast.
The actual modification of the yeast's genetic material happens when the recombinant DNA is integrated into the yeast cell. The uptake of the plasmid into the yeast cell is called transformation. In the laboratory, transformation can be triggered using multiple methods, such as chemical treatment of the yeast cells, or electroporation. Electroporation uses high-voltage pulses to increase the permeability of the cell membranes, which allows foreign DNA to enter the cell. During transformation, not every yeast cell will take up the plasmid, which is why the successfully transformed yeast cells have to be distinguished from the unsuccessful ones. For that purpose, the plasmids used for molecular cloning usually contain an additional selective marker besides the target gene or altered DNA sequence. This marker, which is often an antibiotic resistance gene, allows selective growth of the genetically modified yeast on agar plates that contain the specific antibiotics. In many cases parts of the plasmid DNA of a successful transformant is sequenced to confirm that the correct DNA sequence is present in that particular clone.
Genetic engineering is not limited to yeast or bacteria. The genomes of plants and animals can be altered in a similar way. Many agricultural plants such as corn, canola, or soybeans have already been genetically engineered to improve their performance. Another application of GMOs is in the pharmaceutical industry. Today, many medicines, like insulin for diabetics or certain antibodies, are produced by genetically modified bacteria or plants. Scientists are also looking into the production of other commercially valuable products, such as spider silk, biofuels, or vaccines.
In this project, you will create your own genetically modified yeast. Specifically, you will make Saccharomyces cerevisiae glow by introducing a green fluorescent protein gene (gfp) into its DNA. GFP originates from a jellyfish and it is often used in biotechnology to mark certain cells or as a reporter gene that indicates if a cell expresses a certain protein. In this experiment, you will transform a yeast plasmid that contains the gfp gene into Saccharomyces cerevisiae. You will do this transformation with different strains of yeast. There are many different strains of yeast, including baker's yeast or brewer's yeast (which is used for wine-making or for brewing beer). You will investigate if each of the different strains is able to take up the GFP plasmid during transformation. Do you think you can make all of the yeast strains fluoresce under UV light?
Terms and Concepts
- Saccharomyces cerevisiae
- Model organism
- Genetic engineering
- Molecular cloning
- Genome editing
- Genetically modified organism (GMO)
- Recombinant DNA
- Restriction enzyme
- DNA ligase
- Agar plate
- Green fluorescent protein gene (gfp)
- What is the purpose of model organisms? Can you give examples of different model organism?
- How can you get foreign DNA into a bacteria or yeast cell?
- What is GFP and what is it used for in research?
- Your Genome. (2016). Why use yeast in research?. Retrieved August 31, 2020.
- New England BioLabs. (n.d.). Foundations of Molecular Cloning - Past, Present and Future. Retrieved August 28, 2020.
- Phillips, T. (2008). Genetically modified organisms (GMOs): Transgenic crops and recombinant DNA technology. Nature Education 1(1):213
- MilliporeSigma. (n.d.). Introduction to Yeast Transformation. Retrieved August 31, 2020.
Materials and Equipment
- Genetically Engineer Any Brewing or Baking Yeast to Fluoresce Kit
available from The ODIN.
To get 10% off the purchase price of the kit, use the coupon code "buddies" at checkout.
The kit includes:
- YPD agar (1)
- YPD agar containing G418 antibiotic (1)
- 250-mL glass bottle for pouring plates (1)
- 10-100 μL professional lab-grade variable volume adjustable pipette (1)
- Box 1-200 μL pipette tips (1)
- Petri dishes (14)
- Microcentrifuge tube rack (1)
- Inoculation loops / plate spreader / pairs of nitrile gloves in plastic bag (5)
- Microcentrifuge tubes (25)
- 1.5-mL microfuge tubes containing YPD (5)
- 50-mL centrifuge tube for measuring liquid volume (1)
- Orange film square and blue light (1)
- Packet of Saccharomyces cerevisiae strain (1)
- 1 mL yeast transformation buffer 40% PEG 8000, 200mM LiAc, 0.1mg/mL salmon sperm DNA (1)
- Pre-engineered strain of yeast containing GFP plasmid in an agar stab (1)
- Yeast GFP expression plasmid 100 ng/μL
- Yeast, see the Procedure for suggestions of which types
- Pencil or pen
- Permanent marker
- Lab notebook
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Bacteria are all around us in our daily lives and the vast majority of them are not harmful. However, for maximum safety, all bacterial cultures should always be treated as potential hazards. This means that proper handling, cleanup, and disposal are necessary. Below are a few important safety reminders.
- Keep your nose and mouth away from tubes, pipettes, or other tools that come in contact with bacterial cultures, in order to avoid ingesting or inhaling any bacteria.
- Make sure to wash your hands thoroughly after handling bacteria.
- Proper Disposal of Bacterial Cultures
- Bacterial cultures, plates, and disposables that are used to manipulate the bacteria should be soaked in a 10% bleach solution (1 part bleach to 9 parts water) for 1–2 hours.
- Use caution when handling the bleach, as it can ruin your clothes if spilled, and any disinfectant can be harmful if splashed in your eyes.
- After bleach treatment is completed, these items can be placed in your normal household garbage.
- Cleaning Your Work Area
- At the end of your experiment, use a disinfectant, such as 70% ethanol, a 10% bleach solution, or a commercial antibacterial kitchen/bath cleaning solution, to thoroughly clean any surfaces you have used.
- Be aware of the possible hazards of disinfectants and use them carefully.
For health and safety reasons, science fairs regulate what kinds of biological materials can be used in science fair projects. You should check with your science fair's Scientific Review Committee before starting this experiment to make sure your science fair project complies with all local rules. Many science fairs follow Regeneron International Science and Engineering Fair (ISEF) regulations. For more information, visit these Science Buddies pages: Project Involving Potentially Hazardous Biological Agents and Scientific Review Committee. You can also visit the webpage ISEF Rules & Guidelines directly.
The ODIN kit contains enough materials to do five experiments. It also comes with a kit manual that includes the instructions for how to do the experiment. You can also access the kit manual online. In this experiment, you will test if you can genetically engineer different strains of Saccharomyces cerevisiae to make them fluoresce under blue light.
- When you receive the kit, make sure to store all the perishables in the refrigerator or freezer, as advised in the kit manual.
- Follow the kit instructions to make the agar plates.
- Use three different yeast strains for the next step. Prepare competent yeast cells for each strain, as instructed in the kit manual. Here are some suggestions of yeast strain types you can use.
- Scientists always repeat their experiments to make sure they are reproducible. Because you can only do a limited number of experiments with one kit, duplicate the experiments with the yeast strains that did not come with the kit. You can use the yeast strain provided with the kit as a positive control. This means that the experiment should work with the yeast strain provided. If you have enough resources, you can also purchase a second kit to do more experiments.
- Continue to use all three yeast strains for the transformation step and follow the kit instruction to the end. Make sure to label the agar plates so you know which strain you spread on which agar plate.
- Look at all the different plates through the orange filter under the blue light. Then answer all the following questions.
- Do all three yeast strains glow green? Why do you think this is the case?
- What could be some reasons why one or several yeast strains do not glow?
- Do you see any difference between the positive control strain and the other two strains?
- Can you see a difference in fluorescence intensity for all three strains? Does one strain glow more intensely than another?
- If you do not see the positive control strain (the yeast strain provided in the kit) glow, something went wrong during your experimental procedure. Do not get discouraged if your experiment did not work. Science does not always work on the first try! If you still have enough materials, repeat the experiment and pay attention to every detail in each step.
Ask an Expert
This project explores topics key to Industry, Innovation and Infrastructure: Build resilient infrastructure, promote sustainable industrialization and foster innovation.
- Find out how efficient your transformations were in your experiment. To do this, research how to determine the transformation efficiency and calculate it for all your experiments. Do you see a different transformation efficiency between the different strains?
- Vary the experiment procedure to find out the importance of each step. You could, for example, change the heat shock temperature or duration to test what effect this has on the transformation efficiency. You can also vary the recovery step of the yeast after the transformation. Does your experiment still work if you do not let the yeast cells recover?
- In this project, you genetically engineered different eukaryotic yeast strains. If you want to find out how to genetically modify procaryotic cells, such as Escherichia coli, you can do Science Buddies' project Genetically Modified Organisms: Create Glowing Bacteria!.
- Genetic engineering has led to gene editing, which is a technology that allows us to change and edit genes within organisms at precise locations. CRISPR is an emerging technology that has become an important tool for gene editing. In the CRISPR Gene Editing of Escherichia coli Science Buddies project, you can do your own gene editing experiment using CRISPR.
- Despite the potential benefits and promises of genetic engineering, there are also concerns and risks associated with GMOs that need to be considered. Do some research and find arguments for and against the use of GMOs.
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