‭Bacterial Transformation In‬

‭Action‬

‭Sawyer Wiglesworth, 8‬‭th‬ ‭Grade‬

‭The Lexington School‬

‭Dr. Hurst, Room 49‬

 ‭Table of Contents‬

‭Abstract‬ ‭………………………………..‬ ‭-- 3 --‬

‭Background Research‬ ‭………………………………..‬ ‭--3-5--‬

‭Experiment Details‬ ‭………………………………..‬ ‭--5-6--‬

‭Materials and Procedures‬ ‭………………………………..‬ ‭--6-8--‬

‭ hallenges and Technical‬

C ‭………………………………..‬ ‭--8 --‬
‭Issues‬

‭Experiment Results‬ ‭………………………………..‬ ‭--8 --‬

‭Data Analysis & Discussion‬ ‭………………………………..‬ ‭--9--‬

‭Acknowledgements‬ ‭………………………………..‬ ‭-9-10-‬

 ‭Bibliography‬ ‭………………………………..‬ ‭--11-‬

‭Experiment Notes‬ ‭………………………………..‬ ‭--11--‬

‭Abstract‬

‭In our experiment, we set out to demonstrate bacterial transformation through the release‬

‭of green and purple pigment in two E. coli bacteria strains. No pigment was produced, likely due‬

‭to a failure on our part. Although, from previous experiments, we know that pigment should have‬

‭appeared, and that the purple would have fared better in the K12 strain, whereas the green should‬

‭have fared better in the B-type strain. In modern medicine, bioengineers use bacterial‬

‭transformation to create bacteria which produce insulin. Through a practice not very dissimilar‬

‭from ours, they can save thousands of lives.‬

‭Background Research‬
‭In our science project, we tested whether, in the presence of two different strains‬

‭of E. coli bacteria, two different plasmids would produce different amounts of pigment. We‬

‭hypothesized that the green pigment would be tolerated better by the K12 strain, and tolerated‬

‭worse by the B-type strain, and vice versa for the purple pigment. We chose this topic because it‬

‭would allow us to explore an area of biology that we had never interacted with before:‬
‭bioengineering. Through this project, we hope to gain a better understanding of bacterial‬

‭transformation, a process that is now used to save so many lives.‬

‭In the modern day, bacterial transformation is a well-studied topic by scientists. Bacterial‬

‭transformation is the process whereby plasmids (circular pieces of DNA) undergo ligation, which‬

‭splits them apart for a researcher to insert a piece of DNA into them. This DNA has the gene we‬

‭want the bacteria to replicate. Then, they are heat-shocked, making the bacterial membrane more‬

‭permeable, allowing the plasmid to enter some of the bacteria (the CaCl2 does basically this‬

‭same thing). Plasmids used in this process contain a gene that makes them resistant to a specific‬

‭antibiotic. Thus, after bacteria are placed on an antibiotic plate, those without the plasmid die.‬

‭Bacteria with the plasmid have become plasmid-factories, and they produce even more‬

‭plasmid-containing bacteria.‬

‭Bacterial transformation was first demonstrated by British bacteriologist Frederick‬

‭Griffith. He was trying to determine whether bacteria killed by heat could be used as a‬

‭pneumonia vaccine in mice. In this process, he discovered that nonvirulent pneumonia strains‬

‭could be made virulent by being introduced to a heat-killed virulent strain. He called this his‬

‭“transforming principle,” which was identified as genetic in 1944. Originally, it was thought that‬

‭E. coli was unable to undergo transformation; however, in 1970 it was discovered that it could be‬

‭treated with calcium chloride to become more porous and take up the plasmid.‬

‭Bacterial Transformation is a major portion of DNA cloning, and with DNA cloning, we‬

‭can do incredible things, such as produce insulin for diabetics. Before 1980, insulin was gathered‬

‭from pig and cow pancreases, as the molecule was similar enough to be a substitute. In the‬

‭1980s, scientists discovered how to encode an insulin-producing gene into E. coli, which allowed‬
‭for insulin to be produced on a never-before-seen scale, saving millions of lives. We used E. coli‬

‭in our experiment for the same reason they did: we have a great understanding of E. coli‬

‭compared to other bacteria.‬

‭In the future, I will progress my learning of bacterial transformation further, and maybe‬

‭consider a career as a geneticist or another related profession. Although my project was, in the‬

‭end, inconclusive, I still learned much. When I began, I knew nothing about this process, and‬

‭very little about genetics. Now, I have improved my knowledge to a level I hadn’t thought‬

‭possible before.‬

‭Experiment Details‬

‭Experiment Question‬

‭Will the pPRL plasmid (DNA from‬‭Chromobacterium violacein‬‭) or the pGRN plasmid (DNA from‬
‭ hromobacterium violacein‬‭) produce more pigment in the K12 or B-type E. coli strains.‬
C
 ‭Experiment Hypothesis‬

‭ he K12 strain will tolerate the pGRN plasmid better, and thus produce more pigment, and the‬
T
‭ -type strain will tolerate pPRL better.‬
B

‭Experiment Variables‬

‭Independent Variable‬ ‭The strain of bacteria in which the two plasmids were present.‬

‭Dependent Variable‬ ‭The amount of pigment produced.‬

‭ e would measure the amount of pigment produced by counting the‬

W
‭number of colonies where pigment appeared.‬

‭Controlled Variables‬

‭Same two types of plasmid used for both bacteria/ same species of‬
‭ acteria/ same process used for both strains.‬
b

‭Materials and Procedures‬

 ‭Materials Used‬

1‭ . 2 E. coli bacteria samples (K12 and B-type)‬

‭2. 1 pPRL plasmid DNA‬
‭3. 1 pGRN plasmid DNA‬
‭4. 4 LB-AMP petri dishes‬
‭5. 2 LB petri dishes‬
‭6. 3 50mM calcium chloride, 3 mlb. Nl‬
‭7. 6 lb broth, 3 ml‬
‭8. 4 microcentrifuge tubes, 1.5 ml‬
‭9. 1 sterile inoculating loop‬
‭10. 4 disposable spreaders‬
‭11. 1 graduated cylinder‬
‭12. 1 disposable pipette‬
‭13. 1 ice bath & 42℃ heat bath‬
‭14. 1 incubator‬

‭Procedures‬

‭1. Label 2 small microfuge tubes either "4-1" or "4-2."‬

‭2. Pipette 200ul of CaCl2 solution into each microfuge tube.‬

‭3. Place the tubes on crushed ice.‬

4‭ . Using a sterile pipet tip, toothpick or inoculating loop, scrape a patch of cells off the 4-1 ar 4-2‬
‭petri dish** Avoid scraping up the agar.‬

5‭ . Swirl the cells in the appropriate tube with cold CaCl2 then repeat for the other patch of‬
‭bacteria.‬

‭6. Gently flick and invert the microfuge tubes, then return them to your ice bucket.‬

‭7. Retrieve 2 aliquots of each plasmid for a total of 4 samples (2x pPRL, 2x pGRN).‬

‭8. Label one of the pPRL tubes "4-1" and label the other pPRL tube "4-2."‬
‭9. Label one of the pGRN tubes "4-1" and label the other pGRN tube 4-2."‬

1‭ 0. Flick the tube with the competent "4-1" strain and then pipet 100 µl of the bacteria into the‬
‭tube labeled "PPRL, 4-1." and an additional 100 µl into the tube labeled "PGRN, 4-1.‬

1‭ 1. Flick the tube with the competent "4-2" strain and then pipet 100 µl into the tube labeled‬
‭"PPRL, 4-2" and an additional 100 ul into the tube labeled "pGRN, 4-2"‬

‭12. Incubate the tubes on ice for -5 minutes.‬

‭13. Heat shock the transformation reactions at 42°C for 90 seconds exactly.‬

1‭ 4. Move the tubes to a rack at room temperature and add 0.5 ml LB to each. Close the caps, and‬
‭invert the tubes to mix the contents.‬

1‭ 5. Label the media-side of the LB + amp petri dishes to indicate the strain you've used ("4-1" or‬
‭"4-27 and the DNA you've transformed them with ("PPRL," "PGRN")‬

1‭ 6. Pipet 250 µl of each sample onto the media of the appropriate petri dish. Spread the sample‬
‭evenly across the dish with a sterile spreader. Discard spreader and remainder of transformation‬
‭mix in 10% bleach solution.‬

1‭ 7. Incubate petri dishes, media side up, overnight at 37°C. After the petri dishes have incubated‬
‭overnight, count the colonies in each dish‬

‭Challenges and Technical Issues‬

‭ he kit that we used cost $150, and that cost prohibited me from conducting more trials. In‬
T
‭addition, due to the shipping time, the volatile nature of our samples, and other factors, it would be very‬
‭difficult to perform many trials. Also, during the heat bath, a central step in our process, you must heat‬
‭shock the samples ofr exactly 90 seconds. Any more or any less can damage them. The most logical‬
‭explanation for our inconclusive results was this step.‬

‭Experiment Results‬
‭In our experiment, neither of the E. coli strains succeeded in producing pigment; therefore, our‬
‭ ypothesis was incorrect. Due to various previous experiments by other scientists, it is clear the K12‬
h
‭strain should have tolerated the pPRL far better than the pGRN, and vice versa for the B-type strain,‬
‭proving our hypothesis incorrect in even a conclusive experiment.‬‭Although the experiment was‬
i‭nconclusive, we still learned an incredible amount of information about bacterial transformation. From‬
‭this experiment, I have gained inspiration for a possible career as a geneticist, or as a student of some‬
‭related field.‬

‭Data Analysis and Discussion‬

‭During the heat shock, the bacteria were most likely exposed to heat outside of their livable conditions‬
f‭ or longer than 90 seconds. As cost and shipping were prohibitive, we couldn’t replicate the experiment in‬
‭time. If I were given the opportunity to redo this experiment, I would perform it in a lab setting where‬
‭conditions are more stable and more reliable.‬

‭Acknowledgements‬

‭ hanks to Carolina Industries, a company that produces science projects and experiments for classrooms‬
T
‭across the nation, as well as BioBuilder Inc., a company that brings bioengineering projects to schools,‬
‭and the company that provided my samples and experiment guide. Thanks to my father, who, during the‬
‭experiment, would become a steadfast lab partner, when my actual partner was unable to help. Thanks to‬
‭Noah Webber, my lab partner who, although unable to appear at the science fair, was instrumental in the‬
‭project. Finally, thanks to my current chemistry teacher, Dr. Jason Hurst, for inspiring and supporting me‬
‭throughout the project.‬

‭Work Cited‬
‭●‬ ‭Lederberg, Joshua‬‭(1994).‬‭"The Transformation of Genetics by DNA: An‬

‭Anniversary Celebration of AVERY, MACLEOD and MCCARTY(1944) in‬

‭Anecdotal, Historical and Critical Commentaries on Genetics‬‭"‭.‬‬‭Genetics‬‭.‬‭136‬‭(2):‬

‭423–6.‬‭doi‬‭:‬‭10.1093/genetics/136.2.423‬‭.‬‭PMC‬‭1205797‬‭.‬‭PMID‬‭8150273‬

‭●‬ ‭Griffith F (1928).‬‭"The Significance of Pneumococcal Types"‬‭.‬‭The Journal of‬

‭Hygiene‬‭.‬‭27‬‭(2): 113–59.‬‭doi‬‭:‬‭10.1017/s0022172400031879‬‭.‬‭PMC‬‭2167760‬‭.‬

‭PMID‬‭20474956‬

‭●‬ ‭Khan, S. (n.d.).‬‭Bacterial Transformation & Selection (article)‬‭. Khan Academy.‬

‭https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-cloni‬

‭ng-tutorial/a/bacterial-transformation-selection‬
‭Experiment Notes:‬

‭No pigment has appeared throughout incubation.‬