In this lab you will perform a procedure known as genetic transformation. Remember that a gene is a piece of DNA which provides the instructions for making (codes for) a protein. This protein gives an organism a particular trait. Genetic transformation literally means “change caused by genes,” and involves the insertion of a gene into an organism in order to change the organism’s trait. Genetic transformation is used in many areas of biotechnology. In agriculture, genes coding for traits such as frost, pest, or spoilage resistance can be genetically transformed into plants. In bioremediation, bacteria can be genetically transformed with genes enabling them to digest oil spills. In medicine, diseases caused by defective genes are beginning to be treated by gene therapy; that is, by genetically transforming a sick person’s cells with healthy copies of the defective gene that causes the disease.

You will use a procedure to transform bacteria with a gene that codes for Green Fluorescent Protein (GFP). The real-life source of this gene is the bioluminescent jellyfish Aequorea victoria. Green Fluorescent Protein causes the jellyfish to fluoresce and glow in the dark. Following the transformation procedure, the bacteria express their newly acquired jellyfish gene and produce the fluorescent protein, which causes them to glow a brilliant green color under ultraviolet light.

In this activity, you will learn about the process of moving genes from one organism to another with the aid of a plasmid. In addition to one large chromosome, bacteria naturally contain one or more small circular pieces of DNA called plasmids. Plasmid DNA usually contains genes for one or more traits that may be beneficial to bacterial survival. In nature, bacteria can transfer plasmids back and forth allowing them to share these beneficial genes. This natural mechanism allows bacteria to adapt to new environments. The recent occurrence of bacterial resistance to antibiotics is due to the transmission of plasmids.

Bio-Rad’s unique pGLO plasmid encodes the gene for GFP and a gene for resistance to the antibiotic ampicillin. pGLO also incorporates a special gene regulation system, which can be used to control expression of the fluorescent protein in transformed cells. The gene for GFP can be switched on in transformed cells by adding the sugar arabinose to the cells’ nutrient medium. Selection for cells that have been transformed with pGLO DNA is accomplished by growth on ampillicin plates. Transformed cells will appear white (wild-type phenotype) on plates not containing arabinose, and fluorescent green under UV light when arabinose is included in the nutrient agar medium. You will be provided with the tools and a protocol for performing genetic transformation. For the plasmid to taken in by the E.coli the cell membrane must be competent. It is postulated that the Ca2+ cation of the transformation solution (50 mM CaCl2, pH 6.1) neutralizes the repulsive negative charges of the phosphate backbone of the DNA and the phospholipids of the cell membrane to allow the DNA to enter the cells. The heat shock increases the permeability of the cell membrane to DNA. While the mechanism is not known, the duration of the heat shock is critical and has been optimized for the type of bacteria used and the transformation conditions employed.

The liquid and solid nutrient media referred to as LB nutrient broth and LB nutrient agar are made from an extract of yeast and an enzymatic digest of meat byproducts, which provide a mixture of carbohydrates, amino acids, nucleotides, salts, and vitamins, as nutrients for bacterial growth. Agar, is derived from seaweed. It melts when heated, forms a solid gel when cooled (analogous to Jello-O), and functions to provide a solid support on which to culture bacteria. The 10-min incubation period following the addition of LB nutrient broth allows the cells to recover and to express the ampicillin resistance protein beta-lactamase so that the transformed cells survive on the ampicillin selection plates. The recovery culture can be incubated at room temperature or at 37°C for 1 hr to overnight. This can increase the transformation efficiency by more than 10-fold.

Gene expression in all organisms is carefully regulated to allow adaptation to differing conditions and to prevent wasteful overproduction of unneeded proteins. The genes involved in the breakdown of different food sources are good examples of highly regulated genes. For example, the simple plant sugar arabinose is a source of both energy and carbon for bacteria.The bacterial genes that make digestive enzymes to break down arabinose for food are not expressed when arabinose is not in the environment. But when arabinose is present, these genes are turned on. When the arabinose runs out, the genes are turned off again. Arabinose initiates transcription of these genes by promoting the binding of RNA polymerase. In the genetically engineered pGLO plasmid DNA, some of the genes involved in the breakdown of arabinose have been replaced by the jellyfish gene that codes for GFP. When bacteria that have been transformed with pGLO plasmid DNA are grown in the presence of arabinose, the GFP gene is turned on, and the bacteria glow brilliant green when exposed to UV light.

2. Open the tubes and, using a sterile transfer pipet, transfer 250 μl of transformation solution (CaCl2) into each tube.

3. Place the tubes on ice.

4. Use a sterile loop to pick up 2–4 large colonies of bacteria from your starter plate. Select starter colonies that are "fat" (ie: 1–2 mm in diameter). It is important to takeindividual colonies (not a swab of bacteria from the dense portion of the plate), since the bacteria must be actively growing to achieve high transformation efficiency. Choose only bacterial colonies that are uniformly circular with smooth edges. Pick up the +pGLO tube and immerse the loop into the transformation solution at the bottom of the tube. Spin the loop between your index finger and thumb until the entire colony is dispersed in the transformation solution (with no floating chunks). Place the tube back in the tube rack in the ice. Using a new sterile loop, repeat for the -pGLO tube.

6. Incubate the tubes on ice for 10 min. Make sure to push the tubes all the way down in the rack so the bottom of the tubes stick out and make contact with the ice.

9. Remove the rack containing the tubes from the ice and place on the bench top. Open a tube and, using a new sterile pipet, add 250 μl of LB nutrient broth to the tube and reclose it. Repeat with a new sterile pipet for the other tube. Incubate the tubes for 10 min at room temperature.

10. Gently flick the closed tubes with your finger to mix and resuspend the bacteria. Using anew sterile pipet for each tube, pipet 100 μl of the transformation and control suspensions onto the appropriate nutrient agar plates.

11. Use a new sterile loop for each plate. Spread the suspensions evenly around the surface of the LB nutrient agar by quickly skating the flat surface of a new sterile loop back and forth across the plate surface. DO NOT PRESS TOO DEEP INTO THE AGAR. Uncover one plate at a time and re-cover immediately after spreading the suspension of cells. This will minimize contamination.

12. Stack up your plates and tape them together. Put your group name and class period on the bottom of the stack and place the stack of plates upside down in the 37°C incubator until the next day. The plates are inverted to prevent condensation on the lid which may drip onto the culture and interfere with your results.

Prediction Questions:

1. On which of the plates would you expect to find bacteria most like the original non-transformed E. coli colonies you initially observed? Explain your predictions.

2. If there are any genetically transformed bacterial cells, on which plate(s) would they most likely be located? Explain your predictions.

3. Which plates should be compared to determine if any genetic transformation has occurred? Why?

4. What is meant by a control plate? What purpose does a control serve?

Observe the results you obtained from the transformation lab under normal room lighting. Then turn out the lights and hold the ultraviolet light over the plates.

1. Carefully observe and draw what you see on each of the four plates. Put your drawings in the data table below. Record your data to allow you to compare observations of the“+ pGLO” cells with your observations for the non-transformed E. coli. Write down the following observations for each plate.

2. How much bacterial growth do you see on each plate, relatively speaking?

3. What color are the bacteria?

4. How many bacterial colonies are on each plate (count the spots you see). Observe the colonies through the bottom of the culture plate. Do NOT open the plates. Use a permanent marker to mark each colony as it is counted. If cell growth is too dense to count individual colonies, record "lawn".
 

    Now multiply this fraction by the total mass (µg) of DNA you began with which is the volume (10 µl) X the concentration (.08µg/µl). _____________________

    c. Now calculate the transformation efficiency, which is a/b. ______________________