Incorporating Biotechnology into the Classroom - GENETIC ENGINEERING LESSON PLAN

"Bioengineered DNA was, weight for weight, the most valuable material in the world. A single microscopic bacterium, too small to see with the human eye, but containing the gene for a heart attack enzyme, streptokinase, or for "ice-minus" which prevented frost damage to crops, might be worth 5 billion dollars to the right buyer."

Michael Crichton - Jurassic Park


DNA (deoxyribonucleic acid) is the genetic code responsible for giving organisms certain phenotypes. One of the basic tools of modern biotechnology is DNA splicing, cutting DNA and linking it to other DNA molecules. The basic concept behind genetic engineering is the process of removing a functional DNA fragment - a gene - from one organism and combining it with the DNA of another organism in order to make the protein that the gene codes for. For example, currently some plants are genetically engineered in that they acquire genes for resistance to pests or diseases. Also, in the cases of gene therapy for humans, functional genes can be given to people with non-functional or mutated genes, such as in a genetic disease like cystic fibrosis.

In this laboratory experiment, students gain a hands-on experience with the technique of genetic engineering. A non-pathogenic strain of E.coli bacteria is mixed with a plasmid that contains the Lux gene and a plasmid that contains the GFP gene, respectively. A plasmid is a small, circular piece of DNA typically allowing a cell to become resistant to an antibiotic and allowing the synthesis of some other gene of interest. The Lux gene is a Luciferase gene that has been isolated and purified from a glowing bacteria, Vibrio fischeri, but is similar to the firefly gene responsible for its "glowing in the dark" phenotype. On the other hand, the GFP gene is a Green Fluorescent Protein gene that has been isolated and purified from the bioluminescent (fluorescent) jellyfish, Aequoria victoria.

Transformation occurs when a cell takes up and expresses added DNA. When competent E.coli cells are successfully transformed with these two different plasmids, they acquire an additional trait. The E.coli cells transformed with the pLux will glow in the dark, whereas the E.coli cells transformed with the pGFP will fluoresce under a long wave UV light.

This genetically engineered plant Glows-in-the-Dark!

This laboratory experiment is an excellent opportunity for the issues regarding the current and the future genetic engineering of plants, animals, and eventually HUMANS to be discussed. In the very near future, the Human Genome Project will be completed and therefore, the genes that code for every aspect of a human being will be identified. The impact of this knowledge on society as a whole can be discussed :

  • Should we have the right to genetically engineer ourselves "for the better" or to prevent illnesses?
  • Should we have the right to genetically engineer our children?
  • Will only the rich benefit from this?
  • Will this generate "segregation" in our world - the genetically engineered people and the non-genetically engineered people?

A genetically engineered mouse that Glows-in-the-Dark!


  1. Transform bacterial cells with a plasmid and observe the growth of the bacteria and the genetically engineered phenotypic traits. The plasmids used will include some of the following:
    pLux: allows resistence to ampicillin and allows bacteria to GLOW IN THE DARK.
    pGFP: allows resistence to ampicillin and will make the bacteria fluoresce green under a "blacklight".
  2. Learn about plasmids, genes, antibiotic resistence and phenotypic traits.
  3. Learn about genetic engineering and the impact on agriculture.
  4. Learn about gene therapy.
  5. Learn about the Human Genome Project.
  6. Learn about the future of genetic engineering and its impact on society.


Items needed per class (six groups per class):

  1. Qiagen Maxi-Prep purified plasmid DNA (pLux and pGFP) - six aliquots at 100ng/5ul
  2. Water (six aliquots)
  3. Competent E.coli cells ( 3 X 100 ul. aliquots per group) on ice
  4. LB/AMP (Luria Broth with Ampicillin) plates (3 per group - 1 for pLux, 1 for pGFP, and 1 for Control {no DNA}
  5. 37oC Incubator
  6. Long wave UV lights
  7. Straws
  8. LB (Luria Broth) - 1 ml. aliquots per group
  9. 10-100 ul. capacity micropipettors


Instructor: The instructor will prepare competent bacterial cells. Bacterial cells normally will not take up plasmid DNA unless conditioned to do so. The following procedure will be used to prepare competent cells:

  1. Grow an overnight culture of the bacteria (E.coli) in 2 ml. SOB media
    Prepare SOB as follows:
    20 g bactotryptone
    5 g bacto-yeast extract
    0.5 g NaCl
    0.2 g KCl
    Bring to 1 Liter total volume with water.
    Adjust pH to 7.0 with 5 N NaOH.
    Autoclave solution for 20-30 minutes.

  2. Transfer this 2 ml. culture to a flask and add 125 ml. SOB.
  3. Incubate at 370C for 2 hours with shaking.
  4. Place culture on ice for 10 minutes.
  5. Centrifuge the culture to pellet the cells.
  6. Remove the supernatant and resuspend the cell pellet in 40 ml. cold CCMB buffer.
    Prepare CCMB Buffer as follows:
    11.8 g CaCl2
    4.0 g MnCl2
    2.0 g MgCl2
    0.7 g KCl
    100 ml glycerol
    Adjust pH to 6.4 with HCl
    Bring final volume to 1 Liter with water
    Filter sterilize.

  7. Incubate on ice for 20 minutes.
  8. Centrifuge the culture to pellet the cells.
  9. Remove the supernatant and resuspend the cell pellet in 10 ml. cold CCMB buffer.
  10. Aliquot the cells into 100 ul. volumes and quick freeze in liquid nitrogen or dry-ice ethanol bath.
  11. Store the cells at -800C.
  12. The day that cells are to be used, thaw the cells on ice (for a minimum of 1 hour).

Students : Students will conduct the bacterial transformation as follows: (divide the class into six groups)

  1. Take each plasmid DNA and mix it with 100 ul. competent bacterial cells.
    Tube 1 = 100 ul. bacteria + pLUX (10 ul.; 100ng)
    Tube 2 = 100 ul bacteria + pGFP (10 ul; 100 ng)

  2. Incubate on ice for 15 minutes.
  3. Incubate samples at 420C for 2 minutes.
  4. Add 100 ul. LB broth to each tube and mix.
  5. Incubate at 370C for 10 minutes (hold tubes in clenched fist).
  6. Label LB agar plates containing ampicillin appropriately.
  7. Pipet the contents of the tubes onto the agar plates.
  8. Fold a straw into a triangle shape and use it as a spreader to spread the bacterial over the plates. ***USE A NEW STRAW FOR EACH PLATE.***
  9. Incubate the plates overnight at 350C and the next day at room temperature for pLUX and 350C for pGFP.
    With pLUX, the luciferase gene product is not stable at 370C and therefore, will not glow unless grown for a day at room temperature.
  10. Observe the plates for bacterial growth and phenotype.
    a. For pLUX, turn off the room light and looking for "glowing".
    b. For pGFP, turn off room lights and observe plates using a blacklight.

Other Recipes

  • LB agar plates :
    10 g bactotryptone
    5 g bacto-yeast extract
    10 g NaCl
    Bring volume to 1 Liter with water.
    Add 15 g bacto-agar
    Autoclave 20-30 minutes to sterilize solution.
    Once cooled to around 550C (warm to touch flask), add 1 ml of 100 mg/ml ampicillin (filter-sterilize)
    Mix and pour into petri dishes (approx. 15 ml/dish)
    Let petri dishes sit overnight to allow agar to harden.

  • Plasmid preparation:
    Recommend using Qiagen Maxi-Preps for plasmid purification
    Should give a plasmid stock of 1 mg. plasmid DNA/ml. Dilute to 100ng./5ul.
    Store plasmids at -200C or -800


Record the number of colonies observed on each plate and any new phenotypic characteristics of the genetically engineered bacteria.