Ampicillin Resistance in E. coli During DNA Transformation

Ampicillin Resistance in E. coli During DNA Transformation

 

Abstract

 

The lab “DNA Transformation-Ampicillin Resistance” tested four different variables (“B1 Amp,” “B1 No Amp,” “B2 Amp,” and “B2 No Amp) , to show how a plasmid with a resistance gene to the antibiotic ampicillin can be used to place the resistance gene into an able strand of bacteria. Both microcentrifuge tubes (labeled “B1” and “B2”) contained the E. coli bacteria and calcium chloride solution. Microcentrifuge tube “B1” received a drop of a solution, which has a gene that is resistant to ampicillin. In the results, “B2 Amp” had no bacterial growth, “B1 Amp” had growth in the form of multiple small colonies, and “B2 No Amp” and “B1 No Amp” both had regular bacterial growth. These results explain how DNA transformation works, and how it can be used in medical science.

 

Index Descriptors

 

Ampicillin: A semisynthetic penicillin used to treat various infections.

 

Cell Suspension: Cells in culture in moving or shaking liquid medium, often used to describe suspension cultures of single cells and cell aggregates.

 

E. coli:  A bacillus Escherichia coli;  a bacillus normally found in the human gastrointestinal tract and existing as numerous strains, some of which are responsible for diarrheal diseases. Other strains have been used experimentally in molecular biology.

 

Resistant: impervious to the action of corrosive substances.

 

Nonresistant: Not resistant, especially to a disease or an environmental factor, such as heat or moisture.

 

Transformation: genetic modification of a bacterium by incorporation of free DNA from another ruptured bacterial cell compare.

 

Agar: A moist support medium used to grow bacteria.

 

Background

 

DNA transformation refers to “the uptake and expression of foreign DNA in a living cell. Originally defined as an inherited alteration of the phenotype of the transformed cell, see stable and transient”. This meaning taking DNA from one cell, and introducing it into another cell, that may give different phenotypic (physical) outcomes. In this lab, a plasmid DNA was given to the microcentrifuge tube “B1” which gave it a gene that is resistant to ampicillin. This means that the results should show “B1” having some sort of bacterial growth on the petri dishes. It is apparent that the transformation occurred if the plate labeled “B1 Amp” had growth in the form of colonies, which means that some of the bacterial DNA took up the plasmid DNA that was resistant to ampicillin, therefor making some of the bacteria resistant as well. The bacteria on the “B1” plate will show DNA transformation because the plasmid DNA will give the bacteria DNA a gene for resistance to the ampicillin.

 

Method

 

To complete this experiment, the following supplies will be needed: four petri dishes containing agar, two microcentrifuge tubes labeled B1 and B2, four sterile toothpicks, four sterile paperclips, ice, two pipettes, and access to an incubator (needed to heat the petri dishes to 37°C for 24 hours).

 

PreLab Day 1

 

Step 1: Use a sterile toothpick to add a very small (about the size of this 0) colony of E. coli into two microcentrifuge tubes (the ones labeled B1 and B2) along with two drops of calcium chloride (which helps the bacterial DNA accept the plasmid DNA). Gently mix the E. coli with the calcium chloride solution until it appears milky. Firmly close both microcentrifuge tubes and safely dispose of the toothpicks that were used into a container to be destroyed(not the trash).

 

Step 2: Place the microcentrifuge tubes into a tub of ice and wait approximately fifteen minutes. (Do not freeze the tubes, the chilled calcium chloride within the tubes are the correct conditions for DNA uptake.)

 

Lab Day 2

 

Step 1: Gently finger flick the microcentrifuge tubes to suspend the cells.

 

Step 2: Open the tube labeled “B1” and use the sterile pipette to place one drop of solution from the “P” tube into the “B1” tube. DO NOT add anything to the “B2” tube. (The plasmid DNA from the “P” tube that was added to “B1” has a gene resistance to the antibiotic ampicillin.)

 

Step 3: Place both tubes in ice again for fifteen minutes. (The cells are kept cold to prevent them from growing within the tubes while the plasmids are being absorbed.)

 

Step 4: Take both tubes out of the ice and put them into a 42°C water bath immediately for 90 seconds. (The temperature change causes the cells to readily absorb the plasmid DNA, that was placed into tube B1.)

 

Step 5: Use a sterile pipette to put five drops of a sterile nutrient broth into both tube “B1” and “B2”.  Close the tubes tightly, and mix the solutions by tipping them gently. (The bacteria are now provided nutrients to help them recover from the calcium chloride and heatshock treatments.) 

 

Step 6: Label the bottom of the four petri dishes with “B1 No Amp,” “B2 No Amp,” “B1 Amp,” and “B2 Amp.” Also, be sure to properly identify the person who is doing the experiment.

 

Step 7: Using another sterile pipette, place three drops of cell suspension from the tube labeled “B1” onto the two petri dishes labeled “B1”. Then use a sterile paperclip to gently and evenly spread the suspension across the agar, be sure not to touch the part of the paperclip that comes into contact with the agar, so as not to compromise the containment of the E. coli. Do the same thing for the cell suspension in tube “B2,” onto the petri dishes labeled “B2”.

 

Step 8: Incubate the four petri dishes upside down for at least 24 hours at 37°C.

 

Step 9: Look over the results of the transformation on each of the four petri dishes.

 

 

Results

 

The results of the lab are as follows:

 

            B2 Amp                     B1 Amp                 B1 No Amp                B2 No Amp

 

 

“B2 Amp” (Amp standing for ampicillin) shows no bacterial growth. This is because the bacteria are killed because they had no resistance to the antibiotic ampicillin. The ampicillin that was introduced to the bacteria killed every cell leaving none to reproduce and grow on the petri dish. The plate labeled “B1 Amp” showed some bacterial growth in the form of many little clusters, called colonies. The growth that is shown is from the bacteria that took up plasmids (that were added in step 2), and then became resistant to ampicillin. Since not all of the bacterium were resistant, only some grew, showing the colonies you see in the above photo labeled “B1 Amp”.  On both plates “B1 No Amp” and “B2 No Amp” the bacteria grew normally. The antibiotic was not present; therefore both the resistant and nonresistant bacteria grew normally.

 

 

 

 

Discussion

 

The result of the experiment did prove that the hypothesis was correct. The procedure went as expected, and no unexpected results were encountered. The data that was collected from the results explain how DNA transformation works and supports the hypothesis. Some of variables that were in the experiment could have produced different results, are incorrect temperatures, inaccurate mixing methods, contamination of the petri dishes or the tools (such as the toothpicks, paperclips, or pipettes), or a breach of containment of the E. coli bacteria. The results directly support DNA transformation in that the plate labeled “B1 Amp” grew in colonies, stating that some of the bacterial DNA had the resistance gene and some did not, supporting the hypothesis in that DNA transformation gave the bacteria the ability to grow because of the gene in the plasmid DNA. The experiment went successfully and proves the hypothesis to be correct.

 

Conclusion

 

In this lab, DNA transformation was proved. The results depict a transformation occurring with the bacteria DNA and the plasma DNA where the bacteria DNA receives a gene from the plasmid DNA that is resistant to ampicillin. The plate “B1 Amp” shows growth in colonies where some of the bacteria was resistant and some was not. The bacteria that was not resistant, simply did not receive the resistant gene through transformation.

 

Acknowledgements

 

I would like to thank the Iowa State University for the clearly outlined lab instructions and equipment. I would also like to recognize my father, Alex Hefflefinger, for reading over this lab report and helping me along the way.

 

Literature Cited

 

  • Iowa State University, Office of Biotechnology. “DNA Transformation-Ampicillin Resistance” Student Instructions. December 2011.
  • Dictionary.com, LLC. Copyright © 2011. Definitions and resources. December 2011.
  • Campbell, A. Neil and Reece, B. Jane. “Biology” textbook, AP Edition. Seventh Edition. Copyright 2005. Pearson Education, Inc.  December 2011.  Pages 293-331 (Chapter 16-17).

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