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In our experiment, we are using the heat shock method of genetic transformation to attempt to insert a gene that encodes for a green fluorescent glow into E. coli bacteria. In general, transformation means change. Knowing this, genetic transformation is when an organism incorporates foreign DNA into its own causing a change. (Weedman 2009) The basic premise was discovered by Fred Griffith in 1928, and was further developed by Avery in the 1940s. (Lorenz and Wackernagel 1994) The three physical ways that genetic transformations can be conducted is via projectile bombardment, electroporation, and heat shock. For this experiment, we are using the heat shock method in which we subject the E. coli bacteria to extreme temperature change. In theory, this sudden increase in temperature will cause the cell membrane to become more permeable, allowing the new DNA to enter and become incorporated into the genome. (Weedman 2009) By using this method, we are hoping to transfer the green fluorescent protein (GFP) into the E. coli bacteria. E. coli is bacteria found in the human gut, and it consists of one large ring of DNA and smaller loops of DNA called plasmids. Since bacteria can transfer these plasmids to each other, it allows the bacteria to transfer useful genetic adaptations to each other. (CEPRAP 2001) GFP is a protein that is found in a jellyfish which allows them to glow in the dark. (Weedman 2009) This protein has been widely used in research as a marker protein in order to mark proteins and follow them throughout organisms. (CEPRAP 2001) The plasmid, pGLO, we are injecting to the bacteria contains not only GFP but also resistance to the antibiotic ampicilin. (Weedman 2009) If the bacteria still grows in the presence of ampicilin, then we will know that it has successfully incorporated the plasmid into it's DNA. The plasmid will also include an arabinose operon. In the end, if this experiment is successfully done, our original E. coli bacteria will be ampicilin resistant and fluoresce green in the presence of arabinose and under UV light.
Material and Methods:
We obtained two microcentrifuge tubes and labeled one +pGLO and the other tube -pGLO. We transferred 250 µL of transformation solution into each tube with a micropipetter with a fresh tip. The transformation solution we used contained calcium chloride (CaCl2). Then, we filled a beaker with ice and placed the two tubes on ice. We used a sterile loop to pick up a single colony of bacteria from the starter plate provided by our TA. Once the sterile loop had a colony of bacteria on it we immersed the tube into the -pGLO substance. We spun the loop between our fingers until the entire colony of bacteria has dispersed into the fluid and returned the tube back to the ice bath. We repeated this process for the +pGLO tube with a new sterile loop and returned it to the ice bath when we were done. Next, we added pGLO plasmid DNA, also provided by our TA, to our +pGLO tube and mixed it together. We then incubated the ice tubes on ice for 10 minutes. While the tubes were on ice, we obtained four Luria Broth agar plates and labeled them on the bottom accordingly: 1 LB plate -pGLO, 1 LB/amp plate -pGLO, 1 LB/amp plate +pGLO, and 1 LB/amp/ara plate +pGLO. After 10 minutes on ice, we placed both tubes in one of the floating racks, carried the ice bath beaker with the tubes to the water bath, and place the tubes in a 42°C water bath for exactly 50 seconds. Once the tubes had been in the ice bath for exactly 50 seconds, we removed the tubes and immediately put them back into the ice bath for 2 minutes. After 2 minutes, we removed the tubes and placed them on a dry rack. We used a fresh tip to add 250 µL of LB nutrient broth to the +pGLO tube. Then, we got a new fresh tip and added the same thing to the -pGLO tube. We closed the tubes and incubated them at room temperature from another 10 minutes. When 10 minutes were over, we flicked the tubes to mix the contents. Then, using a fresh tip for each tube we added 100 µL of the transformation (+pGLO) and control (-pGLO) into their appropriately labeled agar plates. After that, we spread the suspensions evenly around the surface of the agar by quickly skating the flat surface of the loop back and forth across the plate. We used a new sterile loop for each of the 4 plates we did this to, and took care not to break the surface of the agar. Then, we disposed the tips and loops into the appropriate autoclave red bag. Finally, we stacked the four plates and taped them together. We wrote our name and lab section number on the stack of plates and placed them upside down in the tub designated for our finished plates. Our TA then stored these for us in the 37°C incubator for 24 hrs.
For our experiment, we were attempting to genetically transform E. coli to contain a green fluorescent protein. We did this by heat shock method, where we inserted the foreign DNA then put it in a higher temperature for 50 seconds in hopes of integrating the foreign DNA into our specimen of E. coli.
The first plate we examined was the +pGLO with LB and ampicillin. On this plate, I estimated that it was about 5% covered with growth of about 20 bacteria colonies. The colonies appeared to be yellow in color and did not glow under the UV light. Next we examined the +pGLO plate that also contained LB, ampicillin and arabinose. This plate had about the same amount of growth as the first. It was also yellow in color, but when we put it up to the UV light we could see that it was glowing green, although it was a little challenging to see. After we examined those transformation plates, we moved on to observe the control plates. The -pGLO with LB and ampicillin contained no growth at all. On the other hand, the -pGLO with just LB was about 80% covered with many E. coli colonies that were white.
My original hypothesis was that the bacteria on the +pGLO LB/amp/ara would be the plate glowed under UV light while the other wouldn't. I also predicted that the +pGLO LB/amp plate would have growth, but wouldn't fluoresce under the UV light. Along with that I predicted the -pGLO with just LB would flourish, while the -pGLO with LB/amp wouldn't grow at all.
Each of my predictions was supported by the experimental evidence. The control plate -pGLO with LB had E. coli present and thriving. This is consistent with what I predicted, and it makes sense because it was the original bacterial with just the food to help it grow. The plate that contained LB and the antibiotic ampicillin with the original E. coli showed now growth at all. This happened because the original E. coli bacteria do not have resistance to the ampicillin antibiotic; therefore it was all killed off.
The transformation plates were used to help us determine if our vector containing the protein and antibiotic were actually accepted into the DNA of the E. coli. The fact that we had bacteria growth on both of the +pGLO dishes suggested that our E. coli did, in fact, incorporate the new DNA into is genome. I can tell this because both of these plates contained ampicillin. We notice by our examination of -pGLO with LB and ampicillin, that the E. coli itself wasn't able to survive in an environment where ampicillin was present. However, with the +pGLO it was able to survive in the presence of ampicillin which indicates that the plasmid was incorporated into the genome of the newly transformed E. coli. In order for the green fluorescent protein to be "turned on" it has to be in the presence of the sugar arabinose. This is why the +pGLO LB/amp didn't fluoresce, while the +pGLO LB/amp/arabinose one did.
According to research I have found, the results I received are consistent with other similar experiments conducted.
We were able to follow the step by step directions without any clear mistakes. If there were small mishaps that did happen that went unnoticed, they did not affect the results of our experiment. One weakness that was immediately evident in our experiment was that we didn't have a large amount of growth on the bacteria plate that contained +pGLO with LB, ampicillin and arabinose. This made it a little hard to detect whether our bacteria was glowing in the UV light. It would have been nice if more bacteria was present so we could clearly see the results. This may have been improved by giving them more time to grow or by transforming a larger amount of bacteria to begin with.
- Lorenz, MG, Wackernagel W. 1994. Bacterial Gene Transfer By Natural Genetic Transformation in the Environment. Microbiological Reviews 58(3): 563-602.
- University of California, Davis. 2008. Bacterial Transformation: Green Fluorescent Protein. http://ceprap.ucdavis.edu/Equipment/Protocols/pGREEN_transformation_08.pdf. November 8, 2009.
- Weedman, Donna. 2009. Life 102: Attributes of Living Systems Lab Manual. 6th ed. Minnesota: Caché House, Inc. p 105-111