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Arismita Guha Ray AP Biology Mr. Resch 11/22/08 Bacteria Transformation Lab Report Introduction Escherichia coli more often known as E. coli is a large and diverse group of bacteria, which in some cases will cause harmful food poisoning to a person. The bacteria can be found in the lower intestines of warm-blooded animals and can be easily transferred through food, especially meats such as beef (Center 2007). The strain of E. coli being used in this experiment though, is harmless, as most strains of the bacteria are. Transformation, conjugation and transduction are the three ways that bacteria can transfer genetic material. In this lab, we explored how E. coli bacteria can transfer genetic material. To do so, bacteria use fragments of DNA called plasmids from the surrounding environment and incorporate them into their genome. Plasmids are small, circular pieces of DNA which allow bacteria to be adapted to their environment by substituting their DNA for the original DNA. In this lab, our purpose was to try to get E. coli bacteria to incorporate this plasmid, along with the antibiotic ampicillin and the Lux operon to make the bacteria glow. An operon is a combination of DNA, a promoter and an operator (Campbell and Reece 2005). This operon specifically codes for luciferase, an enzyme in fireflies which help them glow. Luciferase also has immunity to ampicillin, which kills the E. coli that doesn’t contain the Lux operon, so if the bacterial genome can contain both the ampicillin and the Lux operon, then the bacteria will glow. Ampicillin is an antibiotic that is used to treat infections of the middle ear, bladder, sinuses, kidney and others. It is also used to prevent the growth of certain bacteria, E. coli bacteria being one of them (Health 2008). Since the lux operon codes for enzymes immune to ampicillin and the strain of bacteria being used is susceptible to ampicillin, the strains of bacteria which do not include the Lux operon will die out, leaving the ones with it to grow and therefore causing the bacteria to glow. Normally, high concentrations of calcium are needed for strains of bacteria to pick up plasmids, so extra CaCl2 is added to the bacteria. To further increase the efficiency of transformation, we heat shocked the strains of E. coli. Heat shocking bacteria makes it easier for the plasmid to move through the membrane of the bacteria’s outer cell membrane (Roe). Lab Questions 1. Please describe, at the cellular/molecular level, the precise steps involved in heat shock. That is, how can we force a bacterial cell to take up a plasmid? (The animation from the ch.20 slides might help here. Follow the link in the slides, click on “techniques” and then click on “transferring and storing” at the top of the page.) Answer: Before we could heat shock our bacteria, we had to neutralize the phosphate backbone so it would be possible for the genetic transformation to occur. To do this, we had to add CaCl2 to the bacteria, since there wasn’t a high enough concentration of calcium in the bacteria before to neutralize it. The calcium chloride first ionizes the cell membrane, and then neutralizes it. During heat shock, there is a rapid rise in temperature, since the bacteria was previously in an ice bath in order to stabilize the cell membrane and make it easier for the CaCl2 to neutralize the phosphate backbones. This rise in temperature creates an imbalance on either side of the cell membrane, creating a positively charged current that sends the strands of DNA through. 2. If any of the predictions regarding bacterial growth made in the pre-lab considerations differed from your observed results, please describe them and explain why you believe you obtained these results. Answer: Much of our acquired results differed from our earlier predictions. The reason for this was most probably human error. Most likely, our bacteria were somehow mutated throughout the process of the experiment, making it impossible to achieve our earlier predictions. We predicted that in the agar plate containing just the untransformed bacteria, there would be no glow and the bacteria would grow as normal and we were correct in that prediction. We were also correct when we predicted that the agar plate which contained the untransformed bacteria and the ampicillin would not grow. However, our results for the two other agar plates were wrong. We had originally predicted that in the plate with the transformed bacteria and no ampicillin, some of the bacteria would glow and grow but that did not happen in our result. The agar plate with the transformed bacteria, the ampicillin and the plasmid we had expected would glow but that also did not occur in our result. 3. What are you selecting for in this experiment? (i.e., what allows you to identify which bacteria have taken up the plasmid? Answer: Using our results, we can very easily detect which bacteria have taken up the plasmids. Since the ampicillin will prevent the growth of the bacteria which have not taken up the plasmid, and the lux operon will make every living bacteria glow, we can determine that the bacteria that glow are the ones that have taken up the plasmid. 4. Transformation efficiency is expressed as the number of antibiotic-resistant colonies per μg of plasmid DNA. The object is to determine the mass of plasmid that was spread on the experimental plate and that was, therefore, responsible for the transformants) the number of colonies) observed. Because transformation is limited to only those cells that are competent, increasing the amount of plasmid does not necessarily increase the probability that a cell will be transformed. A sample of competent cells is usually saturated with the addition of a small amount of plasmid, and excess DNA may actually interfere with the transformation process. a. Determine the total mass (in μg) of plasmid used. Remember that you used 10 μL of plasmid at a concentration of 0.005 μg/ μL. Answer: Concentration x volume = mass (0.005 g/L) x (10 L plasmid) = 0.05 micrograms (g). plasmid b. Calculate the total volume of cell suspension prepared. Answer: 250μL of CaCl2 + 5 μL of E. Coli + 10 μL of Lux operon plasmid + 250μL of Luria Broth= 515 μL of cell suspension c. Now calculate the fraction of the total cell suspension that was spread on the plate. Answer: Amount spread/total volume cell suspension = total cell suspension (100 L spread) / (514 L total) = 50/257 = 0.1945525292 d. Determine the mass of plasmid in the cell suspension spread. Answer: Fraction of total cell suspension spread x total mass of plasmid = mass of plasmid in cell suspension spread 50/257) x (0.05 g plasmid) = 5/514 g = 0.0097276265 = 9.7276265 x 10-3 g plasmid e. Determine the # of colonies per μg of plasmid DNA. Express your answer in scientific notation. This is your transformation efficiency. Answer: 1 colony/ (1/103 μg of plasmid) = 1*102 colony/ μg of plasmid 5. What factors might influence transformation efficiency? Explain the effect of each factor that you mention. Answers: There are many factors that might affect the efficiency of transformation. Since the chance of mutation in bacteria is so high, any small human error could cause mutation and alter the results of the experiment. For example, if someone opened the top of the agar plate too high while adding the plasmid, mutation could occur. Also, since the transfer of the bacteria from the ice bath to the heat shock needed to be immediate, any small delay could produce unwanted results. Also, the amounts of the material were very important. If there weren’t enough of the plasmids, the bacteria or the ampicillin, the results would be altered. The same goes for there being too much of the plasmid, ampicillin or bacteria. The last important factor is the temperature of the heat shock. This is very crucial, since the bacteria could die if the temperature was too high, and if it wasn’t high enough, then the heat shock wouldn’t be effective and the DNA wouldn’t be able to be transferred. Conclusion: The purpose of this lab was to transform bacteria to emit a glow. In order to do this, we needed to employ the lux operon, which contained the enzyme luciferase which made fireflies glow. In order for the lux operon to be transformed into the DNA, the plasmid containing it needed to reach the bacteria. All of the bacteria that did not contain the plasmids were killed by the antibiotic, ampicillin. To allow passage through the cell membrane of the bacteria, we used a technique called heat shock and the chemical CaCl2. The control of this experiment was the agar plate that didn’t include the plasmid or the ampicillin, where growth of the bacteria would occur but there would be no glow. Our experiments were the agar plates that included both the plasmids and the ampicillin. We predicted that in the agar plate containing just the untransformed bacteria, there would be no glow and the bacteria would grow as normal and we were correct in that prediction. We were also correct when we predicted that the agar plate which contained the untransformed bacteria and the ampicillin would not grow. However, our results for the two other agar plates were wrong. We had originally predicted that in the plate with the transformed bacteria and no ampicillin, some of the bacteria would glow and grow but that did not happen in our result. The agar plate with the transformed bacteria, the ampicillin and the plasmid we had expected would glow but that also did not occur in our result. Since our group was the only group that got this result, we concluded that the plasmids had not properly reached the bacteria. Works Cited Health and Medicine (2008). Ampicillin. Retrieved November 23, 2008, from Health and Medicine Information Produced by Doctors Web site: http://www.medicinenet Campbell, Neil A., & Reece, Jane B. (2005). Biology. San Francisco: Benjamin Cummings. Center for Disease and Control (2008, March 27). Disease Listing: Escherichia coli General Information. Retrieved November 23, 2008, from Center for Disease Control and Prevention Web site: http://www.cdc.gov/nczved/dfbmd/disease_listing/stec_gi.html#1 Roe, Bruce A. Bacterial Transformation and Transfection. Retrieved November 23, 2008, from Department of Chemistry and Biochemisty at Univerity of Okhlahoma Web site: http://www.genome.ou.edu/protocol_book/protocol_adxF.