Genetic transformation is when a cell takes in DNA from an organism other than its own and expresses it. There are three different ways that this process can occur including: projectile bombardment, electroporation, and heat shock. Heat shock is the method that was used for the in class lab with Escherichia coli. The green fluorescent protein (GFP), originally from a bioluminescent jellyfish, was used along with E. Coli to produce a fluorescent glow. The GFP gene has been used before in heat shock experiments with different types of plants along with GUS (gene from uidA gene) and LUC (firefly gene)(Matsuhara, 2001). The activation of this GFP gene requires the presence of the sugar arabinose. A specifically constructed plasmid, pGLO, used as a vector for transformation, contains the GFP gene along with a gene for resistance to ampicillin. Reason behind the ampicillin resistance gene is for screening for transformed cells. Only cells that took up the pGLO plasmid will survive on the media to which the antibiotic ampicillin had been added.
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Heat Shock is when cells are hit with an abrupt increase in temperature, which increases permeability and therefore, DNA is taken up from the surrounding medium. This method is used in the �translocation of proteins into intracellular compartments� in which the proteins are required to be in a temporary unfolded or partly folded form. To achieve this result, heat shock is administered to the proteins in use and thereby, producing the desired result. (Craig, 1993). Heat shock has also recently been researched and used under the topic of cancer! Heat shock once administered to specific proteins in the body have been found to elicit anti tumor immunities! (Blachere, 1997). I predict that the +pGLO/amp/ara will grow DNA the fastest and will be the only one to glow under UV light because the GFP gene needs the sugar arabinose to activate. I predict that the pGLO must be present as well because it includes the GFP gene itself and the gene resistant to ampicillan and this gene must be present to ensure growth.
Materials and Methods:
First insert 250 micro liters of transformation solution into each tube. �Transformation solution contains calcium chloride (CaCl2), which enhances the permeability of the cell membranes, increasing the competency of the bacterial cells and increasing the efficiency of successful transformation�(Weedman 108). Label one tube -pGLO (lacking the pGLO plasmid) and one +pGLO (containg the pGLO plasmid) then place both tubes in an ice bath. Using a sterile loop pick up a colony of bacteria provided and put one in the +pGLO tube and one in the -pGLO tube. Next obtain a loop of DNA pGLO plasmid, insert it into the +pGLO tube only and mix the pGlo plasmid with the E. Coli fluid and transformation fluid. Place both tubes in ice for 10 minutes. We cool the solutions to lower the temperature in order to make the heat shock more dramatic and more easily achieved. Next get 4 LB (Luria Broth) agar nutrient plates and label them: -pGLO LB/amp (LB for Luria Broth and amp for ampicillin), -pGLO LB, +pGLO LB/amp, and +pGLO LB/amp/ara (ara for the sugar arabinose). After the 10-minute ice bath put the two tubes into the 42 degree Celsius water bath for 50 seconds. After the 50 seconds have passed, place the tubes back on ice for 2 minutes. After those 2 minutes, take the tubes out and place 250 micro liters of LB nutrient broth to the +pGLO tube and then do the same for the �pGLO. Close both the + and � pGlo tubes and let them sit at room temperature for 10 minutes. Finally, pipette 100 micro liters of +pGLO and �pGLO onto the correlating LB nutrient agar plates. Then, using a different sterile loop for each one, evenly spread the solution on the agar plates. The plates will be stored in the 37 degrees Celsius incubator for 24 hours.
When observing the results of the experiment a week later the percentage of agar surface covered by bacterial colonies, number of individual colonies, color of colonies under normal light, and color under UV light was recorded. For the +pGLO LB/amp plate 6% percent was covered and there were around 34 small yellow bacterial colonies that did not glow under UV light. The +pGLO LB/amp/ara plate was only about 3% covered with around 12 colonies of yellow bacteria that glew fluorescent under the UV light. The �pGLO LB/amp grew no colonies while the �pGLO LB plate was covered 97% by one very large yellow colony (or perhaps many colonies all grown together) that did not glow under UV light.
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Genetic transformation of E. Coli through heat shock to inherit the fluorescence of the GFP gene was ultimately achieved. However, not every LB plate in this experiment fluoresced under UV light. My hypothesis was proven correct in that only the +pGLO/amp/ara plate had the characteristic of fluorescence when observed under UV light. As discussed in the introduction to this lab, the GFP gene requires the presence of the sugar Arabinose to be activated. The pGLO plasmid must also be present because it contains the GFP gene (fluorescent gene) itself and the gene resistant for ampicillin (so that bacteria will grow on the plate). The +pGlo/amp/ara plate is the only one that meets all of these requirements and therefore is the only one that had bacteria colonies fluoresce under UV light, showing that it took up the pGLO plasmid.
Blachere, Nathalie E. "Heat Shock Protein?Peptide Complexes, Reconstituted In Vitro, Elicit Peptide-specific Cytotoxic T Lymphocyte Response and Tumor Immunity." JEM 186 (1997): 1315-322. The Journal of Experimental Medicine. Rockefeller University Press, 20 Oct. 1997. Web. 1 Dec. 2009. <http://jem.rupress.org/cgi/content/abstract/186/8/1315>.
Craig, Elizabeth A. "Heat-shock proteins as molecular chaperones." Wiley InterScience 219.1-2 (2005): 11-23. Wiley InterScience. Wiley & Sons Inc., 3 Mar. 2005. Web. 1 Dec. 2009. <http://www3.interscience.wiley.com/journal/119967905/abstract>.
Matsuhara, Shio. "Heat-shock tagging: a simple method for expression and isolation of plant genome DNA flanked by T-DNA insertions." Wiley InterScience 22.1 (2001): 79-86. Wiley InterScience. John Wiley & Sons, Inc., 25 Dec. 2001. Web. 1 Dec. 2009. <http://www3.interscience.wiley.com/cgi-bin/fulltext/119188192/HTMLSTART>.
Weedman, Donna. Life 102; Attributes of Living Systems. Eden Prairie: Cache House, Inc, 2009. Print.