Genetic Transformation in E Coli

Genetic Transformation is the act of changing of DNA in an organism by adding new genes, which may be done in multiple ways. The addition of new genes to DNA could have an almost infinite amount of advantages, ranging from studying the cultures of bacteria that become immune to modern medicine, to making artificial animal proteins. In a CNN article written by Matt Ford, scientists are using genetic transformation to do research on the use of growing animal proteins that the scientists claim will be healthier for the plant and mean less animal cruelty. However, the idea of artificially grow animal protein is still very controversial.  In the experiment performed by our lab, we used the idea of heat shock to genetically transform E coli. Heat shock is the process of exposing the cells to a temporary yet extreme increase in temperature, which temporarily "opens" the membranes of the cells. The purpose of opening these membranes is that the genes that are placed in the surrounding area will slip into the cell and become part of the DNA of that cell. In this experiment, we were testing whether or not the heat therapy opened the membranes of the cells, and therefore attempting to complete genetic transformation.  In the paper "Nonchromosomal Antibiotic Resistance in Bacteria: Genetic Transformation of Escherichia coli by R-Factor DNA" by Stanley N. Cohen, Annie C.Y. Chang, and Leslie Hsu is also an example of this kind of genetic transformation on E Coli. After the E. Coli was exposed to CaCl₂, the E coli did not fully become resistant to antibiotics. The reason was that the E coli also needed the right temperature and conditions for the genes to fully become effective in the E coli. After the E coli was introduced to a heated environment for a short time, and then allowed to change and grow in an incubated environment, the resistance for the antibiotics increased in the E coli. While there have been cases where it was found that heat shock therapy was not necessary to engage the genetic transformation cycle, as shown in the article "One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution", we still used the idea of heat shock therapy for our experiment.

To begin the experiment, we took two microcentrifuge tubes and labeled them +pGLO and -pGLO. Next, taking a micropipette with a clean tip we put 250 microliters of transformation solution into each of our microcentrifuge tubes. We then put ice into a beaker large enough for ice and our two tubes, and put these materials into the beaker. After, a sterile loop was used to take a single colony of bacteria into each of our tubes, using separate loops to keep them sterile and avoid contamination. After obtaining another new sterile loop, we put the loop into a tube marked pGLO plasmid DNA. This loop was then put into the tube labeled +pGLO and mixed. After this, we left both tubes in the ice beaker for at least ten minutes to get them and their contents to a lower temperature. While these are on ice, we obtained 4 Luria Broth (LB) nutrient agar plates from our lab provider; one LB plate, two LB/ampicillin plates, and one LB/ampicillin /arabinose plates were given to us. After the ten minutes were over, both tubes were place in water that was 42 degrees Celsius for 50 seconds. After this warm water treatment, we immediately place the tubes back into the ice beaker. After two minutes in the ice beaker, we removed the tubes from the ice beaker. Using a clean tip for each tube on the micropipette, we added 250 microliters of LB nutrient broth to the +pGLO tube and the -pGLO tube and let mixtures sit for ten minutes. After the ten minutes, we gently flicked the tubes to mix the contents of the tubes. Then, we added 100 microliters of +pGLO to the LB/amp nutrient agar plate, 100 microliters of +pGLO to the LB/amp/are plate, 100 microliters of -pGLO to the LB/amp plate, and 100 microliters of -pGLO to the LB plate. Using a new clean and sterile loop for each plate, spread the mixtures of each plate so that they are mixed up well, while being sure not to press hard into the plate. We then closed the plates with their lids and stacked them on top of each other, putting tape around them to keep them in order. We then placed the plates into an incubator for one week.

In this experiment, we introduced the pGLO plasmid to E. coli bacteria so that the cells were genetically transform a resistance to ampicilin as well as the ability to produce the protein that causes a glow. We used heat shock therapy in order to introduce the pGLO plasmid stored in an incubation unit the bacteria in agar plate containing ampicilin, arabinose and nutrient broth. As a result, the agar plate containing nutrient broth with the bacteria that had not been given the pGLO plasmid had bacteria grow in the plate. The plate containing nutrient broth and ampicilin with the bacteria, which was not given any pGLO, did not have any bacterial growth in the plate. The plate with nutrient broth and ampicilin that had the bacteria that had been given pGLO did grow new bacteria, but it did not glow. The final plate containing nutrient broth, ampicilin and arabinose and the bacteria that had been given pGLO both grew new bacteria and also glowed under the light.

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I stated that I believed that the E. coli bacteria that had been given pGLO would not only grow in the presence of ampicilin, but would also glow in the light when there was also arabinose. The results of the experiment did not disprove my hypothesis since the bacteria that had been given pGLO grew in both of the plates with ampicilin present, and glowed in the plate with arabinose present as well. The results of this experiment were consistent with other similar experiments with the same use of heat therapy on genetic transformation. A prime example is the experiment conducted by Cohen, Chang and Hsu in which the method of heat shock was used to introduce antibiotic resistance to E. coli bacteria (Cohen, Chang, Hsu, 1972). The results of the experiment showed that the introduction of R-factor DNA could genetically transform E. coli bacteria to have certain resistances. This experiment helps support our findings since their procedure and outcomes were very similar to our experiment. A few possible errors that occurred in our experiment could include the fact that the bacteria sat for a week after the first part of the experiment instead of being examined after 24 hours, which may have altered the amount of bacteria that was cultured. Also, it was almost impossible to get two halves of the same colony so it is possible that the two samples of E. coli were not genetically identical. However, we do not believe that our experiment had been sufficiently flawed to cause significant error

Citations:

1. Meat is murder? Well, perhaps not for much longer. By Matt Ford. http://www.cnn.com/2009/TECH/science/08/07/eco.invitro.meat/index.html Accessed 11-11-2009

2. "Nonchromosomal Antibiotic Resistance in Bacteria: Genetic Transformation of Escherichia coli by R-Factor DNA" by Stanley N. Cohen, Annie C.Y. Chang, and Leslie Hsu. http://www.pnas.org/content/69/8/2110.abstract.  Accessed 11-10-2009

3. "One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution" by C T Chung, S L Niemela,, and R H Miller. http://www.pnas.org/content/86/7/2172.abstract accessed 11-10-2009

Donna Weedman, 2009 Life 102 Attributes of Living Systems, Cache House Inc. Eden Prairie, MN