Transformation of Escherichia coli with pUC8 Plasmid AP Biology 5th Hour Melissa Bollman 28 February 2014

Introduction The purpose of this experiment is to attempt to create ampicillin resistant Escherichia coli through the process of transformation. Transformation is a change in genotype or phenotype of a bact6eria by assimilation of external DNA. We attempted this by having the E. coli take up the pUC8 plasmid which has an ampicillin resistant gene in it, which will cause the E. coli to be ampicillin resistant. For this experiment we hypothesized that the E. coli on plate 4 would pick up the pUC8 plasmid, become ampicillin resistant, and then proceed to grow on the plate even though the plate has ampicillin on it. E. coli should also grow on plates 1 and 2 because they lack ampicillin and not grow on plate 3 because there is ampicillin preventing E. coli growth. Methods We conducted this experiment in Sterling Heights, MI on 3 February 2014. This experiment was done between 12:13 P.M and 1:09 P.M. and the temperature of the room was 24C. During the entirety of this experiment, we used sterile technique. To begin this experiment, we obtained two microcentrifuge tubes and marked one tube with +, and the other -. The + tube had the plasmid added to it. Next, we took a sterile graduated syringe and added .250 mL (250l) of ice cold calcium chloride to each tube. We used calcium chloride because it increases competency. When this was completed, we grabbed a starter plate and use a sterile inoculating loop to transfer a large colony of E. coli from the starter plate to each tube of cold calcium chloride. Place the loop into the calcium chloride and twirl rapidly to remove the E. coli from the transfer loop. Using the capillary micropipets and plungers, we added 10 L of the pUC8 plasmid solution with the antibiotic resistant to the + tube. We gently tapped the tube to mix the plasmid into the solution and then incubate both tubes on ice for fifteen minutes. We set the tubes in ice for 15 minutes. While the tubes are on ice we obtained two Luria agar plates and two Luria agar plates with ampicillin. We labeled one of the Luria agar plate without ampicillin with +, and the other -. We did the same with the Luria agar plates with ampicillin. We removed the tubes from ice and immediately placed them in a 42C hot water bath for 80 seconds. The bacterial cells must be heat shocked to increase competency and allow for the plasmid to enter the cells. Remove tubes from the water bath and immediately place them on ice for two minutes. We took the tubes out of the ice bath and use a sterile disposable graduated syringe to add .250 mL (250l) of Luria broth to each tube. (The tubes can now be kept at room temperature). Once the Luria broth is inserted, we gently tapped the tube to mix the solution. Luria broth contains minerals and nutrients for bacteria, acting as their food source. We then .100 mL(100l) of the + solution to the two + plates with a sterile disposable graduated syringe. Using a different sterile disposable graduated syringe, we added .100 mL(100l) of the - solution to the two - plates. Next we used a Bacti-spreader to spread the cells over the entire surface of the Luria agar - plate. We repeated this process on the Luria agar with ampicillin - plate. Using a new Bacti-spreader, we spread the liquid over the entire surface of the Luria agar + plate. We repeated this process on the Luria agar with ampicillin + plate. We allowed the plates sit for five minutes to absorb the liquid. After the five minutes are completed, we placed the plates in a 37C incubator, inverted, for 96 hours. After 96 hours passed, we removed the plate from the incubator. We recorded whether there was growth on each plate.

Results Below is the data collected during this experiment: Growth on Plates after 96 Hours of Incubation

Table 1: The first column in this table shows the number of the plate that we were observing. The second column is the specifics of the plate, which are if they contained the pUC8 plasmid or not and if the plate contained ampicillin or not. Lastly, the third column states if there was bacterial growth in the plate. Discussion We thought that the E. coli would grow on plate 4 because those bacteria should take up the pUC8 and gain penicillin resistance. This hypothesis was inaccurate. According to Table 1, there was no colonies present on plate 4. There are several plausible explanations for why our hypothesis was incorrect. The plasmid may not have been added correctly to the '+' tube, the E. coli may have not transformed in high enough quantities, or the E. coli may not have been spread across plate 4 correctly. We also thought that plate 1 would have growth because there is no ampicillin on that plate and the plate has all the nutrients needed for E. coli growth. The same is true for plate 2. As you can see in Table 1, there was E. coli growth on plates 1 and 2. Concerning plate 3, we thought that plate 3 would not have growth because the E. coli added to this plate did not have ampicillin resistance like that found in the pUC8 plasmid. Also in Table 1, there was no E. coli growth on plate 3. The control plates in this experiment were plates 1 and 3 because the E. coli added to these plates were not mixed with the pUC8 plasmid.