Bacterial Transformation Lab Report

2 ABSTRACT The technique of transforming cells such as bacteria in genetic is pertinent for the improvements of molecular biology. The objectives of this lab are: to perform genetic transformation using the bacterial transformation method, and to observe the results of bacterial transformation in various growth condition. As mentioned above, the methodology used was bacterial transformation protocol. Which is a series of technique used to introduce a foreign plasmid into the bacteria (E. Coli), then the bacteria will multiply within the plasmid. If executed properly, the results should display growth and glow under –pGLO with LB, +pGLO with LB/Amp, +pGLO with LB/Ara, and +pGLO with LB/Ara/Amp. Therefore, leads to the conclusion that bacteria will transform under the right conditions. INTRODUCTION The bacteria being transformed in this lab is Escherichia Coli (E. Coli), which is a rodshaped bacteria that is usually found in the lower intestine of warm-blooded organisms. The process of bacterial transformation revolves mainly around the plasmid needed for bacteria’s growth. Within the plasmid, there are genes such as: the AraC protein which will regulate the Larabinose operons in E. coli, the PBAD promoter that will aid in the transcription of AraC, the Green Fluorescent Protein (GFP) that will glow under UV light, the Bla gene that codes for the enzyme beta-lactamase which provides bacteria with antibiotics, and the Ori gene that will signal for the origin of replication. This report analyzes an experiment conducted in order to transform E. Coli using GFP. The null hypothesis is that the E. Coli bacteria will glow and grow the most under +pGLO with LB/Ara. While the alternative hypothesis is that the E. Coli bacteria will glow, and grow the most under +pGLO with LB/Ara/Amp. Bacterial transformation is crucial for in science because it allows genetically engineered bacteria to produce medicines. Due to that reason, this lab is interesting because it provides a deeper understanding of how the topic works, and the lab might result in an innovative finding about bacterial transformation. MATERIALS/METHODS The procedures of this lab span across several days, the steps are as follow: The first step was to retrieve two micro test tubes and label both with the group’s name, and differentiate one as +pGLO and the other –pGLO. Using a sterile transfer pipette, the next step was to inject 250µl of transformation solution (CaCl) into both tubes and instantly place them on ice for three minutes. Step three is using a sterile loop to transfer a colony of E. coli into the +pGLO tube. Note that the loop was harshly spun in order to disperse the entire colony into the solution. Then the +pGLO tube was placed back into the ice for another three minutes. Step three was then repeated for the –pGLO tube. Once the three minutes is up, a new sterile loop was used to collect pGLO plasmid DNA and placed into the +pGlow tube. It is important that this step only occurred for the +pGlow tube. Both tubes were then placed back on ice to incubate for 10 minutes. While the tubes were incubating, 1 LB plate, 2 LB/Ampicillin plates, 1 LB/Arabinose plate, and 1 LB/Ampicillin/Arabinose plate were obtained and correctly labeled. After the incubation period, the bacteria must go through a “heat shock” technique by placing both tubes into a water bath of 42°C for exactly 50 seconds and back into the ice for another two minutes. Once this is over, another sterile pipette was used to add 250µl of LB nutrient into both tubes, and another 3 incubation period begins for 20 minutes at room temperature. The next step was to mix both tubes evenly and use pipettes to transfer 100µl of the transformation and control suspensions onto the corresponding nutrient agar plates. Note that a new sterile loop was used for each plate and the suspensions were spread around the agar plate using the quadrant method. The last step was to stack all of the plates upside down, place them together and incubate at 37°C for a day. The next day was done by the lab instructor, which was to retrieve plates from incubator. The independent variables were the presence/absence of arabinose and ampicillin on each plate, while the dependent variables were whether E. Coli exhibit growth/glow. The controls were chosen depending on which aspects of the experiment is emphasized. When looking for growth, the positive control was the -pGLO with LB plate because the plate contained nothing to inhibit its growth, and the negative control was the -pGLO with LB/ampicillin, because there were no Beta-Lactamase to protect the bacteria from ampicillin. When looking for evidences of glow, the negative control was the -pGLO with LB plate, because it did not have the DNA to make GFP. The positive control was the +pGLO LB/ampicillin/arabinose plate, there were DNA to make GFP, and the arabinose to activate the GFP gene. RESULTS Relative Growth (+++ or + or -) Fluorescent Under UV (Yes or No) Figure 1a title: Actual Bacterial Transformation Results -pGLO -pGLO +pGLO +pGLO LB LB/Amp LB/Amp LB/Ara Normal Contamination/ No growth (-) Little growth growth (+) no growth (-) (+) No No No No +pGLO LB/Amp/Ara No growth (-) No Figure 1b title: Expected Bacterial Transformation Results -pGLO -pGLO +pGLO +pGLO +pGLO LB LB/Amp LB/Amp LB/Ara LB/Amp/Ara Relative Growth Normal No growth (-) Some growth Lots of growth Lots of (+++ or + or -) growth (+) (+) (++) growth (+++) Fluorescent Yes (but not much Yes, lots of Under UV No No No because of glowing (Yes or No) competition) colony Caption for figure 1: The expected versus the actual relative growth (+++ or + or -) and degree of glow under UV light (Yes or No) for five plates tested under the conditions of -pGLO with LB, -pGLO with LB/Amp, +pGLO with LB/Amp, +pGLO with LB/Ara and +pGLO with LB/Amp/Ara. Figure one describes the results of the experiment and display it in a table. Compared to the expected results, it can be seen that the actual results showed none of the plates glowed, one of the plate contracted a contamination, one of the plate did not grow as expected and three out of the five plates showed no growth. 4 Figure 2 title: Visualization of the Results under UV Light Caption for figure 2: In order from left to right, plate 1 is -pGLO with LB, plate 2 is -pGLO with LB/Amp, plate 3 is +pGLO with LB/Amp, plate 4 is+pGLO with LB/Ara and plate 5 is +pGLO with LB/Amp/Ara. Figure two visually shows the bacteria’s amount of growth and the degree of glow under UV light. The trend can be interpreted as after the contamination from plate 2, none of the plates displayed results as expected. DISCUSSION The data accepted the hypothesis that the E. Coli bacteria will glow, and grow the most under +pGLO with LB/Ara/Amp. This statement is logical because under this plate, there are enough LB for growth, DNA to create GFP, and arabinose are present to activate the GFP gene. During the experiment, there were some possible errors that could affect the results: The first possible error could be the sterilization of the equipment used. Without proper sterilization of the equipment and workplace, germs or other bacteria could have entered the plasmid. A second possible source of error is the temperature of the water. This experiment relied heavily on the “heat shock” technique, if the temperature were not the same as instructed, it could have affected the pGLO tubes. Another possible error is how the plates were handled, if the lids of the plates were not correctly closed, it could have allowed germs to enter the plates. The experiment does not contain any exceptions, however, there seems to be anomalies within the results. As seen in the “Results” section, the actual results were very far from the expected ones. A contamination occurred in the second plate of -pGLO with LB/Amp. The possible errors mentioned above could be responsible for this contamination. This experiment’s results would have reasonably line up with previous studies if not for the contamination. There will be two studies referenced in this discussion: In the first study, researchers tried to mutate bacteria’s DNA using a novel plasmid. The plasmid used in the study contained a correction primer that can produce DNA that are resistant to the antibiotic. The study found that in the absence of ampicillin, the yield of mutated DNA is in the low percentages. However, when ampicillin is present, the researchers observed 60-90% increase in yield of mutated DNA (Lewis and Thompson, 1990). The second experiment observed the functions and purpose of AraC protein, which is often found in the pGLO plasmid. The study concluded that without Arabinose, AraC becomes a repressor and inhibit the transcription of the other genes within the operon. Overall, these referenced studies concluded that in the right conditions, the bacterial transformation lab can yield successful results. In conclusion, the data states that when trying to transform E. coli with GFP, it is best to fully comprehend the functions and purposes of each gene in the plasmid. The data also implies that altering the DNA of cells are possible, this discovery showcases the growth of scientific discoveries.