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Bacterial Transformation Lab Report
General Biology I (BIO 181)
Arizona State University
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Bacterial Transformation of pGLO Plasmid
Introduction
Bacterial transformation is where bacteria are able to send and receive genes amongst one another in a colony of bacteria. The reason why bacteria perform gene transformation between each other is to share a new beneficial trait or phenotype. A gene is a fragment of DNA that has coding, once introduced inside of the bacteria, gives it the ability to make certain proteins that will give it a particular trait or phenotype. In this experiment the pGLO plasmid was used in bacterial transformation. The pGLO plasmid DNA contains three different genes that will give the bacteria certain traits. It contains the GFP gene, which codes for the GFP protein that can make the bacteria glow green fluorescent. Also it contains the bla gene, which codes for the beta lactamase protein that gives it resistance to the antibiotic ampicillin. Lastly it contains a special gene that can produce the AraC protein, which regulates the transcription of the GFP protein. The GFP protein can only be created if arabinose sugar is present, in which the arabinose reacts with the AraC protein that will signal RNA polymerase to transcribe the GFP protein. In this experiment there were five different agar plates, E Coli bacteria, and each plate had a different combination of either having or not having each of the pGLO plasmid, ampicillin, and arabinose. In all agar plates there was also LB broth, which is a nutrient rich medium to help grow the bacteria. These different combinations showed what happens to the bacteria when it does or doesn’t have the pGLO plasmid, ampicillin, or arabinose. The expected outcomes of the experiment is that if the plate doesn’t have the pGLO plasmid, the bacteria would not have the resistance to ampicillin and would die if it contained ampicillin, and could never make the pGLO protein to glow green fluorescent. If it does contain the pGLO plasmid it would be resistant to ampicillin, and has the possibility to create the pGLO protein. If the plate does not contain ampicillin, the protein will not die and will grow. But if it does contain ampicillin it will kill off all bacteria that does not have the pGLO plasmid that contains the resistance to it. If
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it does not contain arabinose the AraC protein can not react to signal for the transcribing of the GFP gene. But if it does contain arabinose the AraC protein will be able to signal the RNA polymerase to transcribe the GFP protein. This experiment shows what is needed for the bacterial transformation of the pGLO plasmid and how one thing can cause the gene transformation to stop. This experiment was performed to test the hypothesis that an agar plate that has +pGLO, +arabinose, and +ampicillin will produce only bacteria that is resistant to ampicillin, and is green fluorescent under uv light. This experiment is relevant to our world, because this shows how gene transformation works similar to how gene transformation works in agriculture that produce foods that are resistant to certain pesticides to allow the food to grow even in the presence of it. (Philips)
Methods and Materials
In this experiment the independent variables are the pGLO plasmid, arabinose, and ampicillin. While the dependent variable is the quantity of green fluorescent bacteria in the agar plate. There is also a positive and negative control. The positive control has all independent variables, while the negative control has none of the independent variables. It is important to have these controls to see how changing the independent variables affect the dependent variable. The experiment was started by taking two test tubes and they were labeled -pGLO and +pGLO. Then 250 microliters of transformation solution (CaCl 2 ) was transferred into each tube using a sterile pipette and was put into a foam tube rack on ice for three minutes. Then using a sterile loop for each tube, E Coli from the starter plate was scooped and mixed in both tubes until dispersed evenly and put back on ice for three more minutes. A new sterile loop was then put into the pGLO plasmid DNA so that a film covered the loop. It was then swooshed into the +pGLO tube only. The tubes were then put on ice for 10 minutes. While the tubes were on ice, five plates were prepared. One plate had only +LB. Two plates had +LB and +Amp. One plate with +LB and +Ara. And finally one plate with +LB, +Amp, +Ara. After the tubes were done in the
Bacterial Transformation Lab Report
Course: General Biology I (BIO 181)
University: Arizona State University
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