Insertion of a Plasmid Containing the Lux Operon into E. Coli through
Days, Periods 1-2
Arigoni and Mr. Resch
24th , 2008
transformation is a process in which DNA is moved into an organism such that
its genotype or genetic makeup is changed (What
is, 2001). Though easier to accomplish on single-cell bacteria than
multi-cellular organisms, this process allows us to change the traits of the
original test organisms as well pass these changes to any future generations.
The purpose of this lab was to transform E. Coli to include a plasmid
containing the Lux operon, making the
this experiment, we used colonies of E. Coli bacteria and a plasmid that
contained both the Lux operon for
luminescence and a gene for ampicillin resistance. Plasmids are small and carry
only a few genes, but can be replicated with relative ease (Kimball, 2008). By
mixing E. Coli bacteria and plasmid in a solution of Calcium Chloride, the
negative charges on the cell wall and DNA phosphate sugar backbone are
neutralized so that the two no longer repel each other. Afterward, the bacteria
and plasmid solution are heat shocked at 42 °C, momentarily giving the DNA more
energy to enter the cell and maximizing the amount of DNA the bacteria can
absorb (Roe, 1997). This allows for some of the bacteria to contain plasmid
DNA. To determine our level of success, we bred transformed and non-transformed
bacteria on agar plates both containing and not containing ampicillin. Because
all transformed DNA contained both the ampicillin resistance gene and the Lux operon, we hypothesized that the
plate that contained both transformed bacteria and ampicillin should contain
all luminescent bacteria and the plate with transformed bacteria and no
ampicillin should contain some luminescent bacteria. Also, we predicted that no
untransformed bacteria would grow on the ampicillin plate and that
untransformed bacteria would grow on the plate with no ampicillin, but it would
not be luminescent.
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?
Before giving a
heat shock to the bacteria and plasmid DNA solution, it is first treated with
Calcium Chloride. This neutralizes the negative charge on both the bacteria
cell wall and the sugar phosphate backbone on the DNA. This effectively
eliminates the repulsion that had previously existed between the DNA and the
bacterial cell wall. Next, the bacteria and plasmid solution is cooled over a
period of time. This slows the movement of both the DNA and the individual
molecules of the phospholipid bilayer of the cell wall. Whereas the phospholipids
had moved too quickly before to allow the DNA to pass through, it has now
slowed down, opening some gaps. To give the DNA the energy that it needs to
pass through these openings, the solution is quickly placed in a hot
environment. This gives the DNA the energy and the speed to enter the cell.
Thus, the bacterial cell takes up a plasmid.
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.
I had originally
thought that the agar plate that contained no ampicillin, but did contain the
transformed bacteria would exhibit growth of both luminescent and
non-luminescent DNA. I made this hypothesis thinking that some of the bacteria
would have been transformed and some of it would not have, since both could
survive in the agar plate. However, there were no luminescent bacteria in this
culture. This could just be because there were not enough luminescent bacteria
to be visible to the naked human eye. Since this plate did not contain
ampicillin, both transformed and non-transformed bacteria could have survived
in it. Since the heat shock procedure did not transform all of the bacteria in
the original solution, not all of the bacteria that was allowed to grow on the
culture was transformed either. In the ampicillin plate, the only bacteria that
reproduced were those that contained the Lux
operon. This produced concentrated clusters of luminescent bacteria. Even with
these clusters, it was very hard to see the bacteria glow in the dark. However,
in the plate without ampicillin, luminescent bacteria was scattered among the
non-transformed bacteria. Therefore when the bacteria reproduced, the
luminescent bacteria might not have been gathered in one spot, making it much
harder to see their glow.
What are you selecting for in this experiment? (i.e.,
what allows you to identify which bacteria have taken up the plasmid?
experiment, we are trying to see how much of the bacteria have taken up the
plasmid. This can be seen in two ways. Since the plasmid contained both the Lux operon and a gene for ampicillin
resistance, either criterion could determine whether the bacteria have been
transformed. If the bacteria survived in an ampicillin environment, this should
mean that the bacteria contains the ampicillin resistance gene and therefore
that it has picked up the plasmid. Also, if the bacteria glows in the dark,
this implies that the bacteria contains the Lux
operon and therefore has also taken up the plasmid.
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.
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.
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.
10 μL plasmid *
(0.005 μg/ μL) = 0.05 μg plasmid
Calculate the total volume of cell suspension prepared.
250 μL CaCl2
+ (approx.) 5 μL E. Coli + 10 μL plasmid DNA + 250 μL Luria Broth = ~515 μL
Now calculate the fraction of the total cell suspension
that was spread on the plate.
(100 μL spread)/(515
μL total) = 20/103 ≈ .194
Determine the mass of plasmid in the cell suspension
20/103 * 0.05 μg
plasmid = 1/103 μg ≈ 9.7 * 10-3 μg plasmid
Determine the # of colonies per μg of plasmid DNA.
Express your answer in scientific notation. This is your transformation
1 colony / (9.7 *
10-3 μg plasmid) = 1.0 * 102 colonies/(μg plasmid)
What factors might influence transformation efficiency?
Explain the effect of each factor that you mention.
can influence transformation efficiency. First, the amount of plasmid DNA and
the amount of bacteria that are exposed to each other can affect how much
bacteria is actually transformed. If there is a lot of plasmid DNA and very
little bacteria, there will be very little bacteria available to be
transformed. In this case, adding more plasmid DNA would do no good. Conversely,
if there is very little plasmid DNA and a lot of bacteria, a lot of the
bacteria will not be transformed, also making the transformation less
effectiveness of the heat shock can affect efficiency. If the variance in
temperatures during the heat shock is not large enough, the DNA will not be
able to cross the cell wall and enter the cell. However, if the temperature of
the hot water bath is too high, the bacteria will die.
that does not directly influence transformation efficiency but rather the
observation of results is how well the bacteria can grow in the agar plates. If
there is not enough ampicillin in the culture dish, not all of the
non-transformed bacteria will die. Therefore, the colonies that do grow will
not all be transformed bacteria. If too much bacteria is introduced into the
plate, there will not be enough nourishment to sustain all of the bacteria and
much of it will die. Also, our group accidentally scraped away some of the agar
from the culture dish. This deprived some of our bacteria with nutrition and
food, which could explain why we did not see many colonies in our dish.
this experiment, we attempted to transform bacteria to include a plasmid with
the Lux operon for luminescence after
the addition of Calcium Chloride and the usage of a heat shock. The transformed
bacteria on an agar plate containing ampicillin was then our experimental
successfully predicted that the transformed bacteria on the ampicillin plate
would be luminescent, that no untransformed bacteria would grow on an
ampicillin plate, and that untransformed bacteria would grow on an
ampicillin-free plate but that it would not be luminescent. However, our
original hypothesis that some of the transformed bacteria that grew on the
ampicillin-free plate would be luminescent was not supported by the experiment.
This could have been because the combination of both luminescent and
non-luminescent bacteria scattered the luminescent bacteria when the bacteria
reproduced. Since the luminescent bacteria was not in one concentrated area, it
would not have been visible to the naked human eye.
(October 2001). What is Genetic Transformation?
Retrieved Nobember 23, 2008 from Ag
West Bio Inc. Web site:
Kimball, John W. (February 24,
2008). Recombinant DNA and Gene Cloning.
Retrieved November 23, 2008 from
Biology Pages Web site: http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RecombinantDNA.html.
Roe, Bruce A. (December 04, 1997). Bacterial Transformation and Transfection. Retrieved November 23, 2008 from University
Advanced Center for
Genome Technology Web site: http://www.genome.ou.edu/protocol_book/protocol_adxF.html.
This web page was produced as an assignment for an AP Biology course at Montgomery High School.