Download File

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
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
Plate 2: 12 / .2 = 60
Avg: about 67 colonies per ug
11 colonies
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.