Abstract

Have you ever heard or read about GMO? GMO stands for genetically modified organisms. Scientists can deliberately modify the DNA of organisms, such as bacteria or plants, to change their properties for a specific purpose. For example, crops can be modified to become more drought- or pest-resistant. Genetic engineering is a very powerful tool in biotechnology that has already found many different applications in agriculture, medicine, and industry. In this project, you will engineer a non-hazardous strain of Escherichia coli bacteria yourself by inserting a fluorescent protein gene (GFP) into their DNA. If you are successful, you will make your bacteria glow under UV light!

Objective

To determine transformation efficiencies dependent on transformation conditions, while genetically modifying Escherichia coli bacteria so they glow under UV light.

Introduction

Imagine if you could change the appearance or function of any living organism in the world. You could create a cat with pink fur or make a tree that grows candy. The possibilities are endless! This is not as impossible as you might think. Scientists have already figured out how to change the genetic code of living organisms. This process is called gene engineering, gene modification, or gene manipulation. You probably know that all living organisms have DNA in their cells that contain the genetic information for how they develop and function. So how can you make changes to this genetic information?

Genetic engineering uses biotechnology to alter the genes of an organism. A gene is a specific DNA sequence that encodes the synthesis of a protein. When a gene is expressed within an organism, the DNA is first copied into RNA in a process called transcription. The RNA is then converted into a protein during another process called translation (Figure 1). Proteins are like little nanomachines that carry out most functions inside a cell. In a human organism, for example, they help digest our food, keep our heart beating, and control how our muscles move.

The key to changing an organism's property or function is thus to alter its genes. You can either modify or delete an existing gene within an organism or you can add a new gene into its DNA. Creating a genetically modified organism (GMO) involves multiple techniques and several steps. Let's have closer look at how bacteria cells can be genetically modified. First, genetic engineers need to 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 bacteria need to be created and introduced into the host cell. The host organism is the bacteria to be modified. DNA molecules that are formed by laboratory methods to create new DNA sequences are called recombinant DNA.

The technique that allows us to create and assemble recombinant DNA molecules is called molecular cloning (Figure 2). 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 bacteria 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 bacteria. They are usually small, separate from the chromosomal DNA of the cell, and often carry genes that provide a benefit for the bacteria, such as antibiotic resistance. For molecular cloning, 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, can be introduced into the bacteria host cell.

The actual modification of the bacteria's genetic material happens when the recombinant DNA is integrated into the bacteria cell. The uptake of the plasmid into the bacteria cell is called transformation. In the laboratory, transformation can be triggered using multiple methods, such as chemical treatment of the bacteria cells or electroporation. Electroporation uses high-voltage pulses to increase the permeability of the bacteria cell membranes, which allows foreign DNA to enter the cell. In bacteria, plasmids can also be transferred from one cell to another via direct cell-to-cell contact. This process is called conjugation. During transformation or conjugation, not every bacteria cell will take up the plasmid, which is why the successfully transformed bacteria 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 us to selectively grow the genetically modified bacteria 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 bacteria. The genomes of other microorganisms, 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, such as insulin for diabetes or certain antibodies, are produced by genetically modified bacteria or plants. Scientists are also looking into its use in the production of other commercially valuable products, such as spider silk, biofuels, and vaccines.

In this project, you will create your own genetically modified bacteria. Specifically, you will make Escherichia coli bacteria glow by introducing a green fluorescent gene (gfp) into their 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 plasmid that contains the gfp gene into Escherichia coli cells. Once the plasmid has been successfully taken up by the E. coli cells, they will fluoresce under UV light. During your experiment, you will vary the transformation conditions to determine how that changes the cells' ability to take up the GFP plasmid. The transformation efficiency will tell you how efficiently your cells took up the plasmid. The transformation efficiency can be determined by the number of successful transformants (glowing bacteria cells or colonies on the selective plate) generated per microgram of plasmid DNA used for the transformation and spread on the selective agar plate. How many glowing bacteria cells do you think you will have on your plates?

Terms and Concepts

• DNA
• Genetic engineering
• Gene
• Transcription
• Protein
• Translation
• Genetically modified organism (GMO)
• Host cell
• Recombinant DNA
• Molecular cloning
• Restriction enzymes
• DNA ligase
• Vector
• Plasmids
• Transformation
• Agar plates
• Escherichia coli
• Green fluorescent gene (gfp)
• Transformation efficiency

Questions

• What is the role of plasmids in molecular cloning?
• How can you increase or decrease the transformation efficiency of a reaction?
• How are genetically modified organisms (GMOs) used today?

Materials and Equipment

• Genetic Designer Starter Kit - Glowing Jellyfish Bacteria, available from The ODIN. To get 10% off the purchase price of the kit, use the coupon code "buddies" at checkout. Read the Procedure to see if you want one or two kits. The kit includes:
  • LB agar (1)
  • LB Kan agar (Kan [100 μg/ml]) (1)
  • 250 mL glass bottle for pouring plates (1)
  • Disposable transfer pipette (9)
  • Petri dishes (7)
  • Inoculation loops (5)
  • Pairs of nitrile gloves in plastic bag (5)
  • Bag of microcentrifuge tubes (1)
  • 1.5-mL microcentrifuge tubes containing LB broth (5)
  • 50-mL centrifuge tube for measuring liquid volume (1)
  • 100 μL bacterial transformation buffer (25 mM CaCl₂, 10% PEG 8000) (5)
  • Orange UV filter sheet and blue light (1)
  • Non-pathogenic E. coli bacteria freeze-dried tube (DH5α) (1)
  • Sterile water tube (1)
  • Jellyfish GFP plasmid freeze-dried tube (5)
• Pencil or pen
• Permanent marker
• Calculator
• Lab notebook

Experimental Procedure

Variations

Careers

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Related Links

• Science Fair Project Guide
• Other Ideas Like This
• Biotechnology Project Ideas
• Genetic Engineering Project Ideas
• My Favorites

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