Jonathan Paul Loomis

November 17, 1998

TA: Heather Emmitte

This laboratory exercise dealt with the resistance bacteria has to a particular antibiotic in their natural state and in a genetically altered state. We were particularly interested in the affects genetic altering had on the bacteria's resistance to the antibiotic because it relates to genetic engineering in plants and animals, and most lately in humans.

The purpose of this laboratory exercise was to determine if bacteria cells that had been infused with plasmid DNA would present a resistance to an antibiotic. In a broader sense, we were trying to determine if one could add specific codes of DNA in the form of plasmids to a cell and alter its fundamental genetic makeup. In the second half of the experiment we were also trying to determine the specific length of the strands we had added to the cells by using the process of electrophoresis.

I had two hypotheses for this particular experiment. The first dealt with weather or not the bacteria would grow in the environments with and without ampicillin. I predicted that the bacteria in the plate that contained no ampicillin would grow without hindrance. I predicted that the transformed bacteria would grow in the presence of ampicillin, but that the bacteria that had not been transformed would not.

My second hypothesis dealt with the length of the plasmid DNA we added to the bacteria and the results of the electrophoresis. I predicted that our length of plasmid would run close to the dimmer of approximately 2100 or 2200 bp on the standard curve.

The following is a list of materials used in this lab:

E. Coli bacteria

Ampicillin

Microfuge tubes

Micropipets

SOC nutrient broth

Ice bath

Shaker machine

Plastic gloves

Dye buffer

Electrophoreses machine

Electrophoreses gel

Lambda DNA cut with the restriction enzyme Hind III

UV light viewing box

Ager plates

We used the TA's handout for the procedure in place of the lab manual. The only change we made from the handout was that we added 400 micoliters of SOC nutrient in step one of part one instead of 200. We also made a few mistakes in the counting of specific times for the various hot and cold baths the bacteria had to go through in steps three and four of part one.

Sorry, due to the horrible problems at the university computer labs this image could not be scanned. It will come in as soon as possible.

This line of best fit shows the length of our particular plasmid DNA. It is roughly 2,300 base pairs in length, when judged by the fact that it traveled 2.9 cm on the electrophoresis gel.

These two agar plates show clearly that the bacteria grew in all conditions except when ampicillin was present and the bacteria had not been transferred. There were a few minor examples of contamination on the plates as well, but these did not seem to affect the other results. The bacteria in the plate without ampicillin produced a bacterial lawn, while the transferred bacteria in the plate with ampicillin grew up into colonies and not a lawn.

This laboratory exercise was really very informative and interesting. I learned a number of things. I learned that in a relatively short period of time and by way of a relatively simple process one can change the fundamental makeup of the DNA of a cell. In this case it was the E. Coli bacteria, but I'm sure that plant and animal cells would be just as simple to transform. I was also interested to find that the DNA plasmid, once inside the cell, had no problem performing its duties of manufacturing enzymes. I would have guessed that the DNA would have had to bind with the other DNA already in the cell in order to perform its tasks.

My hypothesis concerning the ager plates was correct. The bacteria grew just fine in the plate without ampicillin. The bacteria with the plasmid DNA, which was producing the enzyme that counteracted ampicillin, grew fine in the presence of ampicillin while the bacteria without the enzyme did not grow.

My second hypothesis concerning the projected length of the plasmid DNA that was discovered using the process of electrophoresis was also correct. I had predicted that the plasmid would be of a length near the dimmer of 2,100 bp to 2,200 bp and the actual was approximately 2,300 bp in length.

There is very little that went wrong in this experiment. The most notable problem was the presence of some contamination in the ager plates (which is noted in the results), but this contamination did not seem to affect the results in any way. It is possible that the difference between the bacterial lawn in one plate and the bacterial colonies in the other was a result of some sort of contamination, but rather unlikely.

The short pieces of plasmid will travel much faster than the long ones.

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