Introduction: This lab is about genetic transformation which is the ability of a cell to code for proteins not natural to the cell, meaning that foreign DNA to the cell has to make it's way into the cell. Genetic transformation can happen in a multiple ways, and most of them have plasmids carry DNA into the cell (Gillespie et al. 2007). The method used in this lab will be the heat shock method which involves changing the heat of cells quickly to increase the permeability of the cells. It is used because it is not as intrusive to the living bacteria cells as some other methods, and keeps the cell functioning fairly normally(Kurata et al. 2006).It is similar to sweating by a cell, when it gets hot it opens up it's membrane to allow fluid to exit it. When this happens, DNA can be placed outside the cell and can be carried in by carriers known as plasmids.
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A bacterium used in this lab is E. coli. It is used because it reproduces rapidly and is inexpensive. When the plasmids containing foreign DNA enter the bacteria as the membrane becomes more permeable because of the heat shock to the cell, the DNA is able to replicate to RNA and have its proteins produced, giving the bacteria foreign traits. Plasmids do not normally disrupt to workings of the cell and in this case the E. coli during this transformation (Wang et al. 2001).
Materials and Methods: Two microcentrifuge tubes are needed and should be labeled with the group name. One must be marked pGLO+ and the other pGLO-. Attach a new tip to the micropipetter and without touching the tip to anything but the transformation solution, withdraw 250µL and transport it to one tube. Do this again for the other tube. Once each tube contains 250µL of transformation solution, place them both in the ice. Discard the pipette tip.
Use a new loop and without contaminating it by touching anything besides the bacteria, take a colony off of the starter plate with the loop and place the loop in the pGLO + tube. Spin the loop with your fingers in the transformation solution of the tube until the bacteria is entirely mixed in with the solution. Do the same for the pGLO- tube, only with a new loop. Discard the loops once they have been used.
Use a new loop and place it in the pGLO plasmid DNA tube. Cover the loop with the liquid, take it out, and mix it with the pGLO+ solution until evenly mixed. Dispose of the used loop and place the pGLO+ and pGLO- tubes in the ice beaker for ten minutes.
Get the following agar plates from the TA: one LB/ampicillin/arabinose plate, 2 LB/ ampicillin plates, and one LB plate. Mark the bottoms of the agar plates as followed,
Once the two tubes have been on ice for ten minutes, carry them over to a 42°C bath that is already setup. Place the tubes in the floating water racks for exactly fifty seconds. Once the fifty seconds in the water is up, place the two tubes back in the ice beaker for two minutes.
After the two minutes in the ice bath expires, put them both back onto the tube holder on the table. With a new tip for the micropipetter, take 250µL of LB nutrient broth and add it to the pGLO+ tube. Discard the tip, attain a new one, and add the same amount of nutrient broth to the pGLO- tube. Close the tubes and let them sit on the table rack for ten minutes.
Once the ten minutes are up, shake the tubes ensuring the solution is mixed. Use a new tip on the micropipetter to take out 100µL from the pGLO+ tube and transfer it to one of your agar dishes labeled pGLO+. Do the same for the other pGLO+ dish. Discard the tip, get a new one, and take 100µL from the pGLO- tube and transfer it to a pGLO- dish. Do the same for the other pGLO- dish.
With a new loop, spread the liquid across the surface of the agar plate lightly. Use a new loop for each dish and do this for each dish. Dispose of the used loops.
Always on Time
Marked to Standard
Close the agar dishes. Make sure the group's name and lab section are written on each dish in marker, stack the dishes upside-down, and tape them together. Incubate at 37°C for 24 hours.
Results: In this experiment a protein allowing organisms to glow under UV light was transformed using a plasmid into E. Coli bacteria.
After the agar plates were allowed time to fully incubate both of the pGLO+ plates showed a small amount of E. Coli growth, and the pGLO- plate with just Luria broth showed significant growth. The pGLO+ plate with amilcillin and arabinose had two small colonies of E. Coli which grew and were able to glow under UV light. This indicates that the plasmids containing the green fluorescent protein were successfully transformed into the E. Coli bacteria and were able to grow. Obviously the plasmid did not transform at a very high rate into the bacteria because of small amount of colonies on the plate.
Discussion: In this experiment we started out thinking that the only plate that would glow under UV light would be the pGLO+ LB/amp/ara plate because the ampicillan allowed the bacteria to grow and the arabinose allowed the GFP to glow. This turned out to be true that this would be the only plate with E. Coli capable of glowing. The results supported our hypothesis and confirmed the experiment worked.
Although the E. Coli in the pGLO+ LB/amp/ara could glow, there did not turn out to be as much bacteria on the pGLO+ plates as we would have liked. This may be because the plasmids did not transform into the E. Coli completely. The heat shock method was followed precisely as in the directions as far as timing, so this is not as likely as maybe the bacteria that was placed in the pGLO+ tubes was not all alive of healthy. There also could have been an accidental contamination of the pGLO+ tubes, although they were watched carefully during the whole procedure, but it is still possible they may have been contaminated. There are a number of variables that could have affected the growth of our pGLO+ tubes, so it is very difficult to pinpoint what was the exact misstep in the experiment.
It can be concluded that in this experiment, to successfully have the E. Coli bacteria glow, the plasmid must transform into the bacteria successfully and must contain the activator for the glowing protein and the resistance to the antibiotic on the agar plate. This was achieved by my group for this past experiment.
- Wang L., Brock A., Herberich B., Shultz P. 2001. Expanding the Genetic Code of Escherichia Coli. Science Vol. 292: 498-500
- Kurata H, El-Samad H, Iwasaki R, Ohtake H, Doyle JC, et al. (2006) Module-Based Analysis of Robustness Tradeoffs in the Heat Shock Response System. PLoS Comput Biol 2(7): e59. doi:10.1371/journal.pcbi.0020059
- Gillespie JJ, Beier MS, Rahman MS, Ammerman NC, Shallom JM, et al. (2007) Plasmids and Rickettsial Evolution: Insight from Rickettsia felis. PLoS ONE 2(3): e266. doi:10.1371/journal.pone.0000266