Michael Crichton - Jurassic Park
"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."
INTRODUCTION
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 :
A genetically engineered mouse that Glows-in-the-Dark!
OBJECTIVES
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".
MATERIALS NEEDED
Items needed per class (six groups per class):
PROCEDURES
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:
Students : Students will conduct the bacterial transformation as follows: (divide the class into six groups)
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.
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.
Tube 2 = 100 ul bacteria + pGFP (10 ul; 100 ng)
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
RESULTS
Record the number of colonies observed on each plate and any new phenotypic characteristics of the genetically engineered bacteria.