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Transformation Lab Report

 

 

The Insertion of a Plasmid Containing the Lux Operon into E. Coli through Transformation

  By: Jane Wang

 

AP Biology

ABD Days, Periods 1-2

Mr. Arigoni and Mr. Resch

November 24th , 2008




INTRODUCTION     

            Genetic 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 bacteria luminescent.

            In 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.

                       

 

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?

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.

 

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.

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.

 

3.      What are you selecting for in this experiment? (i.e., what allows you to identify which bacteria have taken up the plasmid?

In this 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.

 

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.

10 μL plasmid * (0.005 μg/ μL) = 0.05 μg plasmid

b.      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 cell suspension

c.       Now calculate the fraction of the total cell suspension that was spread on the plate.

(100 μL spread)/(515 μL total) = 20/103 ≈ .194

d.      Determine the mass of plasmid in the cell suspension spread.

20/103 * 0.05 μg plasmid = 1/103 μg ≈ 9.7 * 10-3 μg plasmid

e.       Determine the # of colonies per μg of plasmid DNA. Express your answer in scientific notation. This is your transformation efficiency.

1 colony / (9.7 * 10-3 μg plasmid) = 1.0 * 102 colonies/(μg plasmid)

 

5.      What factors might influence transformation efficiency? Explain the effect of each factor that you mention.

Various factors 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 efficient.

Also, the 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.

Another factor 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.



CONCLUSION

            In 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 group.

            We 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.  





LITERATURE CITED

 

(October 2001). What is Genetic Transformation? Retrieved Nobember 23, 2008 from Ag West Bio Inc. Web site:

    http://www.agwest.sk.ca/publications/infosource/inf_oct01.pdf.

 

Kimball, John W. (February 24, 2008). Recombinant DNA and Gene Cloning. Retrieved November 23, 2008 from Kimball’s

    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

    of Oklahoma's 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.


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Jane Wang,
Nov 23, 2008, 2:31 PM
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