Introduction:
Bacteria are microscopic, single-celled organisms. Their genetic information is encoded in one large chromosome. It can also be found in plasmids which are small circular pieces of DNA that contain important genetic information for the growth of bacteria. In nature, this information is often a gene that encodes a protein that will make the bacteria resistant to an antibiotic. The reason for this protein being made within the bacteria is because of how bacteria usually grow in the same environment as molds and fungi and compete with them for resources. As a result, molds and fungi have evolved to make toxins that kill bacteria, something that is now used as antibiotics in medicine, in order to gain an advantage over the bacteria. Bacteria, in turn, evolved to make proteins that neutralize the toxins.
Bacteria can transfer this genetic information to other bacteria through plasmids. When a bacterium transforms through obtaining genetic information from an external source, the new genes will be incorporated into the plasmid. This experiment deals with the plasmid pFLO which encodes a gene for resistance to an antibiotic named ampicillin that kills the E.coli bacteria. In this experiment, pFLO shall be transferred to four different colonies of E. coli bacteria. Two of the colonies will be grown on plates with ampicillin and Luria Broth which is composition of ingredients used to promote growth of, in this case, the bacteria.
One of these plates will contain plasmid, while the other will not. The other two plates will just contain Luria Broth. Once again, one will contain plasmid and one will not. The research question for this experiment is: What is the difference in the transformation efficiencies between pFLO and pBLU. Before completing this lab, a first trial was done following the same procedure below but instead of pFLO, pBLU was used. It is believed that the transformations efficiencies should be similar due to the fact that both plasmids are similar in nature.
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By using the techniques of genetic engineering scientists are able to modify genetic materials so that a particular gene of interest from one cell can be incorporated into a different cell. It is necessary to obtain a gene to modify genetic material. First a scientist isolates plasmid DNA from bacteria and DNA carrying a gene of interest from cells of another organism, such as an animal. A piece ...
Materials:
1.5 mL of Calcium dichloride (CaCl2)
One bottle of Isopropanol
Paper Towels (Grab as needed)
2 micro centrifuge tubes
Sterile Toothpicks
Permanent Marker (Sharpie)
Micro centrifuge tube rack
2-20 micro pipet including tips
100-1000 micro pipet including tips
Waste bin for micro pipet tips
2 sterile spreaders
10ul of pFLO plasmid
10ul of sterile distilled water
250ul of luria broth (LB)
2 LB Agar plates
2 LB/ampicillin plates
Procedure:
1. Before gathering materials, clean off the workspace that you will be using with a couple squirts of ethanol and a couple of paper towels. This will help make sure that no germs from the table contaminate the experiment 2. Collect the 1.5 mL microfuge tube containing 250uL of CaCl2. When you get these tubes label one so that it says C (control) and the other pFLO. Also label with team name. 3. Put both tubes in the ice bucket
4. Then take a toothpick from the aluminum can and scrape one colony from the provided bacteria sample. 5. Then with that colony, vigorously tap/twirl it against the side of the control tube and make sure that there are no clumps. 6. With the control tube suspend the cells by pipetting repeatedly for a few seconds. 7. Then Put the control tube on ice.
8. Repeat steps 4-7 but this time instead of focusing on the control, use the pFLO. 9. Then with a 2-20 pipett take 10uL of the cherry plasmid and insert it into the PFLO tube. 10. Once the plasmid is inserted mix well by either lightly tapping or flicking the tube. When finished put the tube back on ice. 11. repeat step 9-10 but instead of using a plasmid insert distilled water and focus on the control tube. 12. Now wait for 15 minutes so the bacteria has time to normalize. 13. After the 15 minutes, put the tubes in a “life raft” container in 42 degree Celsius water for 90 Seconds.
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Use a phone or a timer in order to get an accurate measurement. This step must be exact. 14. Then immediately put the tube in the ice bucket for 2 minutes allowing the bacteria to cool again. 15. After the 2 minutes add 250uL to each tube and mix the contents by tapping or flicking. 16. Put the bacteria in 37 degree Celsius water and wait 15 minutes so that the bacteria returns to a normal temperature. 17. Then gather the four plates needed and start plating.
18. For the first plates use LB and LB/Amp and label them with your team name and pFLO 19. On both you will put 100uL of pFLO in the middle of the plate and then use the spreader to gently spread it around. 20. Repeat step 18-19 but instead use the control instead of pFLO 21. Once this is over wait for 5 minutes.
22. Then turn the plates upside and seal them and put them in the incubator at 37 degrees Celsius. 23. Then clean up all materials and repeat step 1 as the very last thing that you do.
Variables:
Controlled: two agar plates,
Responding:
Manipulated:
Data:
Type of Plasmid
Observations
Picture
Amount of Colonies detected
pBLU
No colonies grew on this plate.
It is completely empty with no trace of bacteria.
Colonies = 0
pFLO
There are many different colonies.
Mostly on the edges of the plate, but some are in the center. Putting the plate under the light made it much easier to see the colonies, and could tell they were pFLO colonies because they glowed in the dark
Colonies = 10
Calculations:
Title of Calculation
Work
Answer
Written Explanation
Mass of pFLO
.01ug(concentration) / uL X10ul (amount in microtube)
.1ug
Fraction of Bacteria From Tube
100uL Bacteria suspension /
500uL total volume
.2
Fractional mass of pFLO Plate
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.1ug mass of pFLO/
.2 fraction on plate
.02ug
Transformation Efficiency
10 colonies/
.02ug Fractional mass of pFLO plate
500 Colonies
Analysis and Conclusion:
Well, contrary to what my lab partner and I believed where the transformation efficiency of the two plasmids would be the same due to the fact that they were both plasmids, the results shown from the experiments were completely off. It appears that the pFLO plasmid was able to help the bacteria resist the antibodies trying to kill it while pBLU plasmid didn’t seem to have any effect at all in terms of helping the bacteria as shown through how there’s nothing growing on the pBLU plate while multiple bacteria colonies are growing on the pFLO plate.
Throughout this lab certain errors were made. One human error that occurred was the fact that one or two of the petri dishes weren’t sealed very properly, both of them due to the writer of this lab report, possibly letting in outside bacteria and skewing up the results. For the next time, it may help make results more accurate if that was done better. Well, after looking at the results the question that came to mind was what the difference between the two plasmids was and why did one affect the bacteria more than the other? In technicality, that’s not really a research question though isn’t it? It’s more of something one could look up online.
