pGLO TRANSFORMATION KIT BY PERMATAPINTAR

pGLO TRANSFORMATION KIT

                                     


Genetic transformation is the process by which a gene or genes from one organism are transferred to another organism via an engineered molecule of DNA. If the procedure is successful, the organism is capable of producing the protein encoded by the transformed gene, thus creating a genetic change. Genetic transformation is commonly used in biotechnology. In agriculture, transformation is used to confer genes for pest, frost and spoilage resistance. Transformation of the human insulin gene into bacteria has allowed for production of the protein on a large scale.1 To aid in bioremediation of oil spills, bacteria are transformed with genes that allow them to digest toxic components of the oil.2 The procedure contained in this lab will allow for the transformation of bacteria with a gene from the bioluminescent jellyfish, Aequorea victoria. A successful transformation will result in the expression of the green fluorescent protein (GFP) in the bacteria, causing them to glow bright green under long-wave UV light.


Transformation and Antibiotic Selection: Genetic transformation in this laboratory will be facilitated by using the pGLO plasmid (see below). A plasmid is a circular, self-replicating DNA molecule which can be contained in a bacterial host cell without interfering with the function of the bacterial chromosome. Bacteria are capable, on their own, of randomly acquiring small pieces of DNA from their environment, but the process is inefficient. The transformation protocol in this lab uses a chemical, calcium chloride (CaCl2), plus heat to increase the efficiency of DNA uptake by the bacterial cell.
Even with the chemical transformation procedure, not every bacterial cell will incorporate the pGLO plasmid into the bacteria, not every cell will receive a copy of the plasmid. To isolate only the cells containing the pGLO DNA, the plasmid contains the beta-lactamase gene which encodes for an ampicillin resistance (Ampr) protein. After the transformation, the cells are grown on a solid medium called an agar plate. This medium will contain the antibiotic ampicillin. In the presence of the ampicillin, only the bacteria containing the pGLO plasmid will have the Ampr protein which will break down the antibiotic, and be able to grow. This process is called antibiotic selection.

FIGURE 1




PROCEDURES
It is important to remember that sterile technique is extremely important for this lab. All the materials provided are sterile and proper handling of these supplies and reagents should result in minimal contamination problems.
Lab Station Materials
2 sterile 1.5 mL microtubes
Sharpie pen
Sterile transfer pipets (10)
Sterile CaCl2 transformation solution (600 μL)
Sterile loops (8)
pGLO plasmid DNA
Ice water baths
Foam microtube rack
LB plate (black stripe)
2 LB amp plates (red stripe)
LB amp/ara plate (green stripe)
Water bath @ 42°C
Sterile LB broth (600 μL)
30°- 37°C Incubator (optional)
Lab tape
Microtube rack

Procedure
1. There are 2 sterile 1.5 mL microfuge tubes at the lab station. Label one tube “+pGLO”, the other “-pGLO”.


2. Locate the tube labeled “Transformation Solution”; this contains a solution of CaCl2. With a sterile pipet, aliquot 250 μL transformation solution to both the -pGLO and +pGLO tubes. Place both tubes in an ice water bath.

3. The lab station will contain a starter plate of HB101 bacteria. These bacteria contain only chromosomal DNA. Take notes on how these bacteria look in visible and UV light for comparison with the transformed bacteria later in the lab. The lab station will also include pre-packaged sterile loops (look like a very small soap bubble wand). Remove a sterile loop from the package being careful not to touch the loop on anything outside the bag. Remove the cover from the starter plate and pick a single colony of the HB101 bacteria. Immediately place the loop into the transformation solution in the tube marked “+pGLO” and spin the loop until the entire colony is dispersed into the liquid. Check to be sure there are no bacterial clumps floating in the transformation solution; cell clumps will negatively affect the efficiency of the transformation. Place the tube back in the ice water bath when the cell suspension is complete.

4. Using a new sterile loop, repeat the procedure in Step 3 for the “-pGLO” tube of transformation solution.

5. Inspect the pGLO plasmid solution with the UV lamp provided and note your observations. What do you expect to see at this step? Take the 2-20 μL pipettor, set it to 5 μL. Carefully remove 5 μL of the pGLO plasmid solution and add the DNA into the “+pGLO” cell suspension. Mix the plasmid with the bacterial cells by tapping the tube gently on the bench top. Close the tube and return it to the ice water bath. Why do you not add plasmid DNA to the tube labeled “-pGLO”?

6. Incubate both the “+pGLO” and the “-pGLO” tubes in the ice water bath for 10 min. Make sure the bottom of the tube is pushed through the foam rack and is in contact with the ice water.

7. While the tubes are incubating on ice, label the 4 agar plates at your lab station as follows:

Be sure to label the bottom of the plates (the portion containing the agar). It is very important to label each plate correctly! Remember, the color code on the side of the agar plates indicates which medium already contains the ampicillin for antibiotic selection and arabinose for GFP expression. It will be confusing to analyze the results if you have mislabeled the plates!

8. To transform the plasmid DNA into the bacteria, the cells must undergo a heat shock. This is performed by removing the foam rack from the ice water bath and placing it rapidly in a 42°C water bath. Incubate the tubes at 42°C for exactly 50 seconds. Again, it is important that the tubes are pushed down in the rack so that the bottom of the tubes have optimal contact with the 42°C water. After the heat shock, immediately place the foam rack back in the ice water and incubate for a further 2 min.

9. Remove the foam rack from the ice water bath and place the tubes in the microtube rack on the bench. Add 250 μL of LB broth to each tube, close the cap and gently tap the “+pGLO” and “-pGLO” tube to mix the contents. Incubate the tubes for 10 min at room temperature. This step of the procedure allows the cells to recover from the heat shock treatment before performing the next part of the experiment. It also lets the cells that have acquired a pGLO plasmid begin to express the β-lactamase protein (for ampicillin resistance) before the cells are placed on plates that contain ampicillin.

10. Using a new sterile pipet for each tube, pipet 100 μL of the transformation and control suspensions onto the appropriate plates.

11. Spread the suspensions evenly around the agar plate by quickly sliding the flat loop surface back and forth across the plate surface. Turning the plate in a circular motion with your fingers while swishing the loop back and forth aids in spreading the bacterial suspensions evenly. Do not press down too firmly or you will gouge the surface of the agar plate. This can complicate both the growth and analysis of the bacteria on the plates. Remember to use a new sterile loop for each plate! Let the plates sit on the bench for 2-3 minutes to allow the suspensions to soak into the plate.

12. Stack the plates and tape them together. Label the tape with the group name and class period, if necessary. Turn the plates upside down (with agar at top of plate) and place in a 37°C incubator overnight. If an incubator is not available, the plates may be grown on the bench top for 2-3 days.

Extraction of bacteria from the petri dish

Present you- Teens of pGLO!