Abstract

Can 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!

Objective

To genetically engineer different strains of yeast to make them fluoresce.

Introduction

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.

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 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

• Eukaryote
• Saccharomyces cerevisiae
• Model organism
• Genetic engineering
• Molecular cloning
• Genome editing
• Genetically modified organism (GMO)
• Recombinant DNA
• Restriction enzyme
• DNA ligase
• Vector
• Plasmid
• Transformation
• Agar plate
• Green fluorescent protein gene (gfp)

Questions

• 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?

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

Experimental Procedure

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Variations

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Related Links

• Science Fair Project Guide
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• Genetic Engineering Project Ideas
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