If I heat shock E. Coli for 60 seconds, at 42 Degrees Celsius, then it will have a higher transformation efficacy than if I had shocked it for 30 seconds, because I think that the lipid bilayer will not be disrupted enough to allow plasmids to enter the bacteria. Since heat shocking disrupts a bacterias membrane since it is hotter than the bacterias ideal temperature, I believe that if I heat shocked it for 60 seconds, it would be better than 30 seconds.

A plasmid is a circular piece of DNA that is often found in bacteria. The plasmid I have is a one that contains a antibiotic resistance gene (Kanamycin) and a GFP protein gene. 

The reason it needs Kanamycin resistance is because we are growing the bacteria on a agar plate with Kanamycin. This in theory should lead to no other bacteria on the plate except for the bacteria that have the Kanamycin resistance gene & GFP gene. It also means that any of the bacteria that have not absorbed the plasmids die also. This leads to a good selection of bacteria. 

Heat shocking is when you shock a bacteria to a heat it is not very comfortable inside. For example, I heat shocked E.coli at 42℃ since it is a temperature E.coli isn’t comfortable in, however won’t die inside. Heat shocking leads to a heat shock response. The membrane of the bacteria starts to fluctuate and this leads to a gap in the lipid layer. Which allows for plasmids (the DNA) to enter the bacteria. After, we let it settle for a couple of hours so that the plasmids settle into the bacteria. After the incubation, we should be able to plate the bacteria since the plasmids have setteled in.

After plating the newly transformed bacteria they should reveal some glow-in-the-dark characteristics.

Manipulated/Independent: The heat shocking time, 30 seconds or 60 seconds

Controlled: The heat of the water, temperature and time of incubation, amount of plasmids

Responding/Dependent: The amount of transformed colonies

 

PROCEDURE:

 

Part 1

Preparing Agar Plates:

 

1. Add LB Agar media to a bottle.

 

2. Add 150mL of water to the bottle with LB Agar media.

 

3. Heat the agar in the microwave at 15 second intervals until almost boiling.

 

4. Once the LB Agar media looks clear, cool until warm

 

5. Carefully remove the lid of the plates and pour agar to cover half the plate.

 

6. Cool agar for about 2 hours.

 

7. Use an inoculating loop to streak bacteria.

 

8. Store plate agar at room temperature for 48 hours. 

 

Part 2

Transforming The Bacteria:

 

1. Pick a colony, and add it to the transformation mix. 

 

2. Store in the fridge for 30 minutes

 

3. Add plasmids into the mix

 

4. Prepare beaker with water at 42℃ using a thermometer

 

5. Split the mix into two microcentrifuge tubes

 

6. Heat the first tube for 30 seconds.

 

7. Heat the second tube for 60 seconds.

 

8. Leave at room temperature for more than 4 hours.

 

9. Prepare Kanamycin LB Agar plates (refer to Part 1)

 

10. Streak “transformed” bacteria and store plate at room temperature for 48 hours.

Using a fluorescent microscope, I observed that 60 seconds transformed some bacteria. 30 seconds did not transform bacteria. I believe that the reason why was because the lipid bilayer was not disturbed enough due to the short period of heat shocking time.

 

1. GFP-negative E. Coli with nucleic stain
2. GFP-positive E. Coli with nucleic stain

 

A plasmid is a circular piece of DNA that is often found in bacteria. The plasmid I have is a one that contains a antibiotic resistance gene (Kanamycin) and a GFP protein gene.
 

The reason it needs Kanamycin resistance is because we are growing the bacteria on a agar plate with Kanamycin. This in theory should lead to no other bacteria on the plate except for our bacteria that have the Kanamycin resistance gene & GFP gene. It also means that any of our bacteria that have not absorbed the plasmids die also. This leads to a good selection of bacteria.

Heat shocking is when you shock a bacteria to a heat it is not very comfortable inside. For example, I heat shocked E.coli at 42℃ since it is a temperature E.coli isn’t comfortable in, however won’t die inside. Heat shocking leads to a heat shock response. The membrane of the bacteria starts to fluctuate and this leads to a gap in the lipid layer. Which allows for plasmids (the DNA) to enter the bacteria. After, we let it settle for a couple of hours so that the plasmids settle into the bacteria.

 

 

60 seconds is better than 30 seconds. I believe that a longer duration like 90 seconds would be more optimal because it will allow the membrane to be disrupted for longer, and more plasmids will be able to enter the bacteria. For 30 seconds, it did not transform the bacteria as shown in observations. This is likely because the lipid bilayer didn't open due to the short time. Overall a longer time such as 60 - 90 seconds for transforming bacteria via heat shocking is better.

Making glow-in-the-dark bacteria is very useful in the lab setting. For example, we can put the bacteria in a mouse and track where the bacteria goes. The thing that matters is the idea of genetically modifying an organism. We are able to modify bacteria so that they can:

 

1) Consume plastic or 2) Destroy cancer cells.

 

Plastic eating bacteria is a promising solution to degrade plastic without any impact to the environment. These bacteria produce enzymes that are able to degrade plastic. One example is when in 2016, Japanese researchers discovered a naturally plastic eating bacteria called Ideonella sakaiensis. This bacteria is able to use enzymes to break down bacteria. Our only problem as of now is making a large quantity of these bacteria. If we obtain the plasmids from Ideonella sakaiensis we can insert them into a different bacteria using the same heat shocking technique I used in my experiment, then we can make large amounts of plastic eating bacteria. Scientists are implementing this right now. 

 

For cancer-eating bacteria, they are naturally absorbed into the thriving tumor environment. Because some bacteria are anaerobic they survive very well in the low-oxygen/hypoxic environment of a tumor. One such case was when Korean researchers injected genetically modified Salmonella into rats with colon cancer. The Salmonella was modified so that it released a protein called Flagellin B, which made immune cells more aggressive. The control rats that didn’t get this treatment eventually died. Eventually in the near future, we may be able to save many lives by using bacterial therapy in cancer patients.

 

-The Variables Were Incorrect:

-60 seconds/30 seconds was not an ideal time for bacterial transformation

-42 C is too hot/cold for bacterial transformation

 

-The bacteria is dead

-I accidentally let in foreign bacteria into the agar plates (I limited this as much as possible)

-The kanamycin in the agar plate doesn't work.

1. Price, Michael. Scientists Turn Food Poisoning Microbe into Powerful Cancer Fighter, science.org, 8 Feb. 2017, www.science.org/content/article/scientists-turn-food-poisoning-microbe-powerful-cancer-fighter. 
2. Zimmer, Carl. “New Weapons Against Cancer: Millions of Bacteria Programmed to Kill.” The New York Times, The New York Times, 3 July 2019, www.nytimes.com/2019/07/03/science/cancer-bacteria-immune-system.html. 
3. Lohner, Svenja. "Genetically Modified Organisms: Create Glowing Bacteria!" Science Buddies, 12 Oct. 2023, https://www.sciencebuddies.org/science-fair-projects/project-ideas/BioChem_p049/biotechnology-techniques/genetic-engineering-glowing-bacteria-transformation-efficiency. Accessed 13 Feb. 2024.
4. Rommasi, Foad. “Bacterial-Based Methods for Cancer Treatment: What We Know and Where We Are.” Oncology and Therapy, U.S. National Library of Medicine, June 2022, www.ncbi.nlm.nih.gov/pmc/articles/PMC9098760/#:~:text=Escherichia%20coli%20Nissle,clearing%20them%20from%20the%20body
5. “Plastic-Eating Bacteria Turn Waste into Useful Starting Materials for Other Products.” American Chemical Society, American Chemical Society, www.acs.org/pressroom/presspacs/2023/november/plastic-eating-bacteria-turn-waste-into-useful-starting-materials-for-other-products.html#:~:text=Now%2C%20researchers%20in%20ACS%20Central,nylon%20materials%2C%20drugs%20and%20fragrances. Accessed 24 Feb. 2024. 
6. “Plastic-Eating Bacteria: Nature’s Recyclers | Let’s Talk Science.” Plastic-Eating Bacteria: Nature’s Recyclers, letstalkscience, letstalkscience.ca/educational-resources/backgrounders/plastic-eating-bacteria-natures-recyclers. Accessed 26 Feb. 2024. 
7. Public Health England. “Health Matters: Antimicrobial Resistance.” GOV.UK, 10 Dec. 2015, www.gov.uk/government/publications/health-matters-antimicrobial-resistance/health-matters-antimicrobial-resistance. ‌
8. Biorender. “BioRender.” BioRender, biorender-marketing-site, 2022, biorender.com/.

Canada, Public Health Agency of. “Government of Canada.” Canada.Ca, / Gouvernement du Canada, 17 Nov. 2021, www.canada.ca/en/public-health/services/antibiotic-antimicrobial-resistance/about-antibiotic-resistance.html..

I am grateful to my mom for supporting me in getting all the materials during my science fair journey. She provided aid with storage, materials, and ideas. I am grateful to my science tutor as well since she helped take microscopic pictures of my bacteria. This is an opportunity I normally would not have been able to have since it is a special type of microscope. Last but not least, this experiment would not have been possible without The Odin since it provided me with a transformation mix, and GFP plasmids for my bacteria, which are materials difficult to obtain in any typical store.