Download Arismita Guha Ray

Arismita Guha Ray
AP Biology
Mr. Resch
11/22/08
Bacteria Transformation Lab Report
Introduction
Escherichia coli more often known as E. coli is a large and diverse group of bacteria,
which in some cases will cause harmful food poisoning to a person. The bacteria can be found in
the lower intestines of warm-blooded animals and can be easily transferred through food,
especially meats such as beef (Center 2007). The strain of E. coli being used in this experiment
though, is harmless, as most strains of the bacteria are.
Transformation, conjugation and transduction are the three ways that bacteria can transfer
genetic material. In this lab, we explored how E. coli bacteria can transfer genetic material. To
do so, bacteria use fragments of DNA called plasmids from the surrounding environment and
incorporate them into their genome. Plasmids are small, circular pieces of DNA which allow
bacteria to be adapted to their environment by substituting their DNA for the original DNA. In
this lab, our purpose was to try to get E. coli bacteria to incorporate this plasmid, along with the
antibiotic ampicillin and the Lux operon to make the bacteria glow. An operon is a combination
of DNA, a promoter and an operator (Campbell and Reece 2005). This operon specifically codes
for luciferase, an enzyme in fireflies which help them glow. Luciferase also has immunity to
ampicillin, which kills the E. coli that doesn’t contain the Lux operon, so if the bacterial genome
can contain both the ampicillin and the Lux operon, then the bacteria will glow.
Ampicillin is an antibiotic that is used to treat infections of the middle ear, bladder,
sinuses, kidney and others. It is also used to prevent the growth of certain bacteria, E. coli
bacteria being one of them (Health 2008). Since the lux operon codes for enzymes immune to
ampicillin and the strain of bacteria being used is susceptible to ampicillin, the strains of bacteria
which do not include the Lux operon will die out, leaving the ones with it to grow and therefore
causing the bacteria to glow.
Normally, high concentrations of calcium are needed for strains of bacteria to pick up
plasmids, so extra CaCl2 is added to the bacteria. To further increase the efficiency of
transformation, we heat shocked the strains of E. coli. Heat shocking bacteria makes it easier for
the plasmid to move through the membrane of the bacteria’s outer cell membrane (Roe).
Lab Questions
1. Please describe, at the cellular/molecular level, the precise steps involved in heat shock.
That is, how can we force a bacterial cell to take up a plasmid? (The animation from the
ch.20 slides might help here. Follow the link in the slides, click on “techniques” and then
click on “transferring and storing” at the top of the page.)
Answer: Before we could heat shock our bacteria, we had to neutralize the phosphate
backbone so it would be possible for the genetic transformation to occur. To do this, we
had to add CaCl2 to the bacteria, since there wasn’t a high enough concentration of
calcium in the bacteria before to neutralize it. The calcium chloride first ionizes the cell
membrane, and then neutralizes it. During heat shock, there is a rapid rise in temperature,
since the bacteria was previously in an ice bath in order to stabilize the cell membrane and
make it easier for the CaCl2 to neutralize the phosphate backbones. This rise in
temperature creates an imbalance on either side of the cell membrane, creating a positively
charged current that sends the strands of DNA through.
2. If any of the predictions regarding bacterial growth made in the pre-lab considerations
differed from your observed results, please describe them and explain why you believe
you obtained these results.
Answer: Much of our acquired results differed from our earlier predictions. The reason for
this was most probably human error. Most likely, our bacteria were somehow mutated
throughout the process of the experiment, making it impossible to achieve our earlier
predictions. We predicted that in the agar plate containing just the untransformed
bacteria, there would be no glow and the bacteria would grow as normal and we were
correct in that prediction. We were also correct when we predicted that the agar plate
which contained the untransformed bacteria and the ampicillin would not grow. However,
our results for the two other agar plates were wrong. We had originally predicted that in
the plate with the transformed bacteria and no ampicillin, some of the bacteria would glow
and grow but that did not happen in our result. The agar plate with the transformed
bacteria, the ampicillin and the plasmid we had expected would glow but that also did not
occur in our result.
3. What are you selecting for in this experiment? (i.e., what allows you to identify which
bacteria have taken up the plasmid?
Answer: Using our results, we can very easily detect which bacteria have taken up the
plasmids. Since the ampicillin will prevent the growth of the bacteria which have not taken
up the plasmid, and the lux operon will make every living bacteria glow, we can determine
that the bacteria that glow are the ones that have taken up the plasmid.
4. Transformation efficiency is expressed as the number of antibiotic-resistant colonies per
μg of plasmid DNA. The object is to determine the mass of plasmid that was spread on
the experimental plate and that was, therefore, responsible for the transformants) the
number of colonies) observed.
Because transformation is limited to only those cells that are competent, increasing the
amount of plasmid does not necessarily increase the probability that a cell will be
transformed. A sample of competent cells is usually saturated with the addition of a small
amount of plasmid, and excess DNA may actually interfere with the transformation
process.
a. Determine the total mass (in μg) of plasmid used. Remember that you used 10 μL
of plasmid at a concentration of 0.005 μg/ μL.
Answer: Concentration x volume = mass
(0.005 g/L) x (10 L plasmid) = 0.05 micrograms (g). plasmid
b. Calculate the total volume of cell suspension prepared.
Answer: 250μL of CaCl2 + 5 μL of E. Coli + 10 μL of Lux operon plasmid + 250μL
of Luria Broth= 515 μL of cell suspension
c. Now calculate the fraction of the total cell suspension that was spread on the
plate.
Answer: Amount spread/total volume cell suspension = total cell suspension
(100 L spread) / (514 L total) = 50/257 = 0.1945525292
d. Determine the mass of plasmid in the cell suspension spread.
Answer: Fraction of total cell suspension spread x total mass of plasmid = mass of plasmid
in cell suspension spread
50/257) x (0.05 g plasmid) = 5/514 g = 0.0097276265 = 9.7276265 x 10-3 g plasmid
e. Determine the # of colonies per μg of plasmid DNA. Express your answer in
scientific notation. This is your transformation efficiency.
Answer: 1 colony/ (1/103 μg of plasmid) = 1*102 colony/ μg of plasmid
5. What factors might influence transformation efficiency? Explain the effect of each factor
that you mention.
Answers: There are many factors that might affect the efficiency of transformation. Since
the chance of mutation in bacteria is so high, any small human error could cause mutation
and alter the results of the experiment. For example, if someone opened the top of the agar
plate too high while adding the plasmid, mutation could occur. Also, since the transfer of
the bacteria from the ice bath to the heat shock needed to be immediate, any small delay
could produce unwanted results. Also, the amounts of the material were very important. If
there weren’t enough of the plasmids, the bacteria or the ampicillin, the results would be
altered. The same goes for there being too much of the plasmid, ampicillin or bacteria. The
last important factor is the temperature of the heat shock. This is very crucial, since the
bacteria could die if the temperature was too high, and if it wasn’t high enough, then the
heat shock wouldn’t be effective and the DNA wouldn’t be able to be transferred.
Conclusion:
The purpose of this lab was to transform bacteria to emit a glow. In order to do this, we
needed to employ the lux operon, which contained the enzyme luciferase which made fireflies
glow. In order for the lux operon to be transformed into the DNA, the plasmid containing it
needed to reach the bacteria. All of the bacteria that did not contain the plasmids were killed by
the antibiotic, ampicillin. To allow passage through the cell membrane of the bacteria, we used a
technique called heat shock and the chemical CaCl2. The control of this experiment was the agar
plate that didn’t include the plasmid or the ampicillin, where growth of the bacteria would occur
but there would be no glow. Our experiments were the agar plates that included both the
plasmids and the ampicillin.
We predicted that in the agar plate containing just the untransformed bacteria, there
would be no glow and the bacteria would grow as normal and we were correct in that prediction.
We were also correct when we predicted that the agar plate which contained the untransformed
bacteria and the ampicillin would not grow. However, our results for the two other agar plates
were wrong. We had originally predicted that in the plate with the transformed bacteria and no
ampicillin, some of the bacteria would glow and grow but that did not happen in our result. The
agar plate with the transformed bacteria, the ampicillin and the plasmid we had expected would
glow but that also did not occur in our result. Since our group was the only group that got this
result, we concluded that the plasmids had not properly reached the bacteria.
Works Cited
Health and Medicine (2008). Ampicillin. Retrieved November 23, 2008, from Health and
Medicine Information Produced by Doctors Web site: http://www.medicinenet
Campbell, Neil A., & Reece, Jane B. (2005). Biology. San Francisco: Benjamin Cummings.
Center for Disease and Control (2008, March 27). Disease Listing: Escherichia coli General
Information. Retrieved November 23, 2008, from Center for Disease Control and
Prevention Web site: http://www.cdc.gov/nczved/dfbmd/disease_listing/stec_gi.html#1
Roe, Bruce A. Bacterial Transformation and Transfection. Retrieved November 23, 2008, from
Department of Chemistry and Biochemisty at Univerity of Okhlahoma Web site:
http://www.genome.ou.edu/protocol_book/protocol_adxF.