Morgan Fox
AP Lab 6: Transformation of E. Coli with pUC8
Question: To what degree will bacteria grow that has been chemically and thermally treated to overcome the effects of ampicillin?
Hypothesis: Bacteria that have been chemically and thermally treated to withstand ampicillin will grow until it has filled its confined space (in this experiment, the Luria agar plate).
Objectives: Investigate transformation as a mechanism of genetic exchange by creating competent cells by chemically and thermally treating E. Coli cells.
Variables: Independent- Presence of pUC8
Dependent- # of Bacteria Colonies
Control- To each tube: 0.25 mL of calcium chloride (at 42 degrees Celsius) added,
0.25 mL of Luria broth added, Incubation on ice for 15 minutes, heat shocked at 42 degree Celsius for 60-90 seconds. For each plate: absorption of liquid into plates for 5 minutes, incubation at 37 degrees Celsius.
Materials: 2 Luria agar plates, 2 Luria agar plates with ampicillin, 2 Microcentrifuge tubes, 1
Inoculating loop, 2 Bacti-spreaders, 4 Sterile graduated syringes, 1 Capillary micropipette with plunger, Calcium chloride, Incubator, Luria broth, Plasmid pUC8,
Waterbath, Starter plate of E. Coli , Thermometer
Procedures: 1. Obtain 2 microcentrifuge tubes and mark on tube “+”, the other “-“. The “+” tube will have the plasmid added to it.
2. Using a sterile graduated syringe, ass 0.25 mL(250 pico meters) ice cold calcium chloride to each tube.
3. Obtain a starter plate. Use a sterile inoculating loop to transfer a large colony of bacteria from the starter plate to each tube of cold calcium chloride. Be sure not to transfer any agar to the tube.
4. To remove the bacteria from the transfer loop, and break them apart, place the loop into the calcium chloride and twirl rapidly. Dispose of the loop according to your instructor.
5. Using the provided capillary micropipets and plungers add 10 picometers of the plasmid pUC8 solution with the antibiotic resistant gene to the “+” tube.
6. Gently tap the tube with your finger to mix the plasmid into the solution.
7. Incubate both tubes on ice for 15 minutes.
8. While the tubes are incubating, obtain two Luria agar plates and two Luria agar plates with ampicillin. Label one Luria agar plate “+”, the other “-“. Do the same for the Luria agar plates with ampicillin. Be sure to label all four plates with your group name.
9. The bacterial cells must be heat shocked to allow the plasmid to enter the cells.
Remove the tubes from ice and immediately place in a 42 degree Celsius hot waterbath for 60-90 seconds.

10. Remove the tubes from the 42 degree Celsius waterbath and immediately place on ice for two minutes.
11. Remove the tubes from the ice bath and add 0.25mL of room temperature Luria broth to each tube using a sterile disposable graduated syringe. Gently tap the tube with your finger to mix the solution. The tubes may now be kept at room temperature.
12. Add 0.1mL of the “+” solution to the two “+” plates with a sterile disposable graduated syringe. Add 0.1mL of the “-“ solution to the two “-‘ plates with a different sterile disposable graduated syringe.
13. Using a sterile Bacti-spreader, spread the cells over the entire surface of the
Luria agar “-“ plate. Then, using the same Bacti-spreader, spread the liquid on the
Luria agar with ampicillin “-“ plate
14. Using a new Bacti-spreader, repeat the procedure for both of the “+” plate.
Spread the liquid on the Luria agar “+” plate first followed by the Luria agar with ampicillin “+” plate. Dispose of the Bacti-spreaders according to your instructor.
15. Let the plates sit for five minutes to absorb the liquid. Place the plates in a 37 degree Celsius incubator, inverted, overnight. If an incubator is not available, invert the plates and allow them to incubate for 24-48 hours.
16. The next day, remove the plates from the incubator. Count and record the number of colonies on each plate. If the bacteria has grown over the entire surface so that the individual colonies cannot be distinguished, write “lawn”. Record your results in the Analysis section on the next page.
Data: see attached sheet (A)
Analysis: The data recorded on the attached sheet shows that thirty-five colonies of transformed E. Coli bacteria grew on the Luria agar plate without ampicillin, a lawn (colonies of bacteria too dense to count) of non-transformed bacteria grew on the control plate without ampicillin, fifty-five colonies of transformed bacteria grew on the plate with ampicillin, and twelve colonies of non-transformed bacteria grew on the control plate with ampicillin.
The two plates without ampicillin, a form of penicillin that can kill many forms of bacteria, both showed growth. On these two plates, it did not matter if the cells had been transformed or not, as there was no antibiotic to restrict bacteria growth.
On the two plates with ampicillin, however, only transformed bacteria (bacteria that have incorporated an ampicillin resistant gene into their own DNA) should have grown. Of these two plates, the one with fifty-five colonies of bacteria had started with transformed bacteria; therefore it makes sense that this plate had so much growth (it does not have a lawn, however, because only those bacteria that were actually transformed were able to grow, those that did not take up the donor bacterium’s DNA could not have developed into ampicillin resistant bacteria and would not have been present). The second control plate, however, had ampicillin and was not supposed to contain any transformed bacteria, so there should not have been any growth. The twelve colonies counted on this plate are likely the result of a different bacterium that had gotten into the plate from its surroundings

(i.e. those working on it may have breathed on it, touched it, it may have been exposed to particles in the air capable of growth, etc.) or the ampicillin in the
Luria agar may have been defective. For the purpose on the intent of this experiment, disregard the incorrect data of the Luria agar with ampicillin-, as this growth was likely due to human error.
Conclusion: My hypothesis should be rejected, as my initial presumption of bacteria growth in the presence of ampicillin was somewhat incorrect. We know that in transformation, DNA is released from its cell into its surroundings. Other recipient cells are then able to take that DNA up into themselves and take on qualities of the donor cell. In this experiment, as we transformed some of the E.
Coli cells, they gained an ampicillin resistant gene that would allow them to grow even when ampicillin was present. It is true that only bacterium that have been chemically and thermally treated (i.e. heat shocked, submerged in ice baths) to withstand ampicillin will grow on Luria agar with ampicillin, but those bacteria will not necessarily grow to the point where they cover the entire surface of the petri dish. Perhaps this is due to the fact that, even though the transformed E. Coli can grow in spite of ampicillin, it is not easy (or as easy as growing on a plate without ampicillin). This experiment, keeping in mind the necessity of rejecting the data of growth of non-transformed cells in the presence of ampicillin, proves that chemically and thermally treated bacteria will grow quite a bit, no matter if ampicillin is present or not.

Questions:
1. Based on your experimental results, did transformation occur? Why or why not?
My results clearly show that transformation did, in fact, occur. Looking at the data from the
Luria agar with ampicillin +, we see that fifty-five colonies of bacteria grew. However, ampicillin was present, and the bacteria E. Coli (the bacteria we used) does not possess a natural system for transformation, and alone cannot overcome ampicillin. These bacteria, however, had been thermally and chemically treated, allowing them to take up the ampicillin resistant gene released from the starting bacteria and become transformed. Thus, transformation occurred.
2. What other methods can be used to verify that transformation occurred? Explain.
In addition to using growth as a method to verify that transformation occurred, we can also use
UV Fluorescence. Since the bacteriophages we injected into the bacteria glow under UV light, we know that if the bacteria grown fluoresces under such a light, that they did indeed take up the
DNA of the donor bacteria and, by definition of transformation, were transformed.
3. Transformation is one type of genetic exchange among bacteria. Research another type of genetic exchange that occurs in bacteria and briefly describe or draw the mechanism by which exchange occurs.
Conjugation is another type of genetic exchange among bacteria. This process is like a form of sexual reproduction for bacteria (certain algae and fungi). Genetic information is exchanged through a temporary fusion by migration of a plasmid from on e bacterium (donor) to another
(recipient).
4. Your expected transformation results for each of the four plates are listed below. Briefly explain why the listed results occurred or did not occur.
Luria “-“: bacterial lawn- without ampicillin present, the bacteria did not need to be transformed to be able to grow on a normal petri dish, and they therefore covered most of the dish

Luria “+”: bacterial lawn- our data showed only thirty-five colonies of bacteria grew on this plate, but it should have been a lawn because many of the bacteria may have been transformed, and those that were not would still be able to grow on the plate in the absence of ampicillin
Luria w/amp “-“: no growth- our data had an error as we recorded twelve colonies of bacteria had grown, but from what we know about bacteria and transformation, E. Coli growth should have been impossible as ampicillin was present, and the bacteria in this dish did not have the gene that would allow them to resist the ampicillin
Luria w/amp “+”: several colonies- these bacteria underwent transformation, but not all of the bacteria transformed into resistant bacteria, so only those that had become resistant were able to grow as ampicillin was present, keeping a lawn from appearing
5. You repeated the experiment and examined te results the next day, below is that your plates looked like when you checked them. Explain what may or may not have occurred.
Luria “-“ Luria “+” Luria w/amp “-“ Luria w/amp “+”
As there was nothing to restrict the growth on the Luria “-“ and “+” plates (no ampicillin present), the bacteria were immeadiately able to grow into lawns. In the case of the two plates on the right, however, the day after there would not have been any visible growth as the Luria w/amp “-“ should not ever grow as the bacteria were not transformed, and the the Luria w/amp
“+” has a sort of battle between the ampicillin resistant bacteria, the non-resistant bacteria, and the ampicillin, before the resistant bacteria will be able to grow.
6. You performed a tranformation and obtained the results on the three plates shown below. You added 10 ug of plasmid at a concentration of 0.005 ug.ul to cellts suspended in
300 ul of CaCl2. After your heat-shock step, 200 ul of Luria broth was added to each reaction. After the experiment was complete, you placed 200 ul of the final solution on each plate and incubated. Calculate the transformation efficiency of each reaction and then calculate the average transformation effciency for all of your trials.
17 colonies 12 colonies 11 colonies
Total mass of plasmid used: 10ul x .005ug/ul = .5ug
Total volume of suspension: 10ul + 300ul + 200ul = 510ul
Fraction of suspension put on plate: 200ul / 510ul = .4ul
Total mass of plasmid in fraction: .5ug x .4ul = .2ug
Number of colonies per ug of plasmid:
Plate 1: 17 / .2ug = 85
Avg: about 67 colonies per ug
Plate 2: 12 / .2 = 60 Plate 3: 11 / .2 = 55

7. Which of the following statements is/are NOT true about bacteria transformation and why? b) It would be incorrect to claim that transformation may only be performed using plasmid DNA containing antibiotic-resistant genes (R factors), because transformation can occur in cases such as gaining the ability to break down an artificial galactoside. e) It is false that cells must be first treated artificially in the laboratory before they are capable of undergoing transformation. This holds true for some cells, such as E. coli , but a significant number of bacteria are capable of undergoing natural transformation, and become competent under ordinary conditions.
8. Using an external source, such as the internet or your school library, research the history of transformation and fill in the missing information in the following paragraphs. A list of terms to be used is included (not all of the terms will be used and some may be used more than once).
The phenomenon of transformation was first discovered in 1928 by Frederick Griffith . In his now famous experiment, he injected mice with living non-pathogenic cells of the bacterium
Streptococcus pneumonia and with heat-killed pathogenic of the same bacterium. The results displayed the properties of living pathogenic cells. This led him to conclude that the living nonpathogenic cells must have been altered in some way by the material from the heat-killed pathogenic cells. Though the exact substance causing the change in the cells was unknown at the time, he proposed that some sort of transforming principle caused the genetic exchange when the two types of cells were combined.
Years later, in the early 1940’s, the work of Oswald Avery , along with several colleagues, demonstrated exactly what the substance was that transformed the cells. By isolating specific components from the heat-killed pathogenic cells an exposing each component individually to the living nonpathogenic cells, it was demonstrated the only material that could cause transformation of the cells was DNA . Their work was met with much skepticism initially because until that point it had been assumed that protein was the source of genetic material.
However, repetition of the experiment and the work of several others proved conclusively that
DNA is indeed the transforming substance, as well as the source of genetic material.

Abstract
I counted the growth of transformed and non-transformed E. Coli in the presence and absence of ampicillin. I did this to determine to what degree bacteria will grow that have been chemically and thermally treated to overcome ampicillin. I found that if ampicillin is present, so must be ampicillin resistant bacteria, else there will be no growth. When ampicillin is absent, however, it goes without saying that bacteria (transformed or not) will have the ability to grow.
This means that we can literally share DNA from one organism to another, allowing it to take on qualities of the donor organism, and transformation can be used as this mechanism for gene exchange.