Laboratory Experiment 6: Molecular Biology for AP Biology
This experiment deals with material from molecular genetics. This is the kind of experiment that can make you feel like a biotech junkie. Here, you use plasmids to move DNA from one cell to another cell—transformation. You get to play with restriction enzymes, e-coli (Escherichia coli—eww), and gel electrophoresis.
For a quick review on molecular genetics, refer to the following concepts:
- Structure, Replication, and Transcription of DNA for AP Biology
- Structure, Process, and Translation of RNA for AP Biology
- Gene Expression and Genetics for AP Biology
Full understanding of this experiment requires a basic knowledge of:
- What vectors are and how they are made.
- What gel electrophoresis is and how it works.
- What a restriction enzyme is and why it is so important to the field of biotechnology.
Okay, I'll tell you now … e-coli (usually abbreviated E. coli in the scientific literature) is a bacteria that is present in everyone's intestinal tract. It grows in the laboratory as well and contains extra chromosmal DNA circles called plasmids. This experiment deals with the process of transformation: the uptake of foreign DNA from the surrounding environment. This is made possible by the presence of proteins on the surface of cells that snag pieces of DNA from around the cell; these DNA pieces are from closely related species.
The goal of this experiment is to take a bacterial strain that has ampicillin resistance, and transform the gene for this resistance to a strain that dies when exposed to ampicillin. After attempting to successfully transform the bacteria, the experimenter can check to see if it was successful by growing the potentially transformed bacteria on a plate containing ampicillin. If it grows as if all is well, the transformation has succeeded. If nothing grows, something has gone wrong …
Part 1: Attempting the Transformation
The first part of this experiment is the attempted transformation. The student adds a colony of E. coli to each of two test tubes. In one tube she adds a solution that contains a plasmid that carries the ampicillin-resistance gene; the other tube receives no such plasmid. The waiting game follows, and after 15 minutes on ice, the two tubes are quickly heated in an effort to shock the cells into taking in the foreign DNA from the plasmid. The tubes are returned to ice and the colonies spread out on an agar plate. They are sent to the incubator to sleep for the night and grow on the plate.
Results from Part 1 (Attempting the Transformation)
Four plates are created: two with ampicillin and two without. The bacteria from both test tubes should happily grow on the plates lacking ampicillin. The ampicillin-coated plate that is spread with bacteria from the nontransformed tube is bare—there is, indeed, no growth. The ampicillin-coated plate that is spread with bacteria from the attempted-transformation tube shows growth … it may not be the greatest growth ever seen, but it is growth. This means that some of the E. coli originally susceptible to ampicillin have picked up the resistance gene from the surrounding plasmid and is transformed.
Important point to take from this part of the experiment: "How in the world does transformation work?" Restriction enzymes are added, which cut the DNA at a particular sequence and open the DNA so that it can be inserted into another such region in the main E. coli chromosome, which is treated with the same restriction enzyme. If the opened DNA from the plasmid happens to find and attach to DNA of the E. coli that is added to the tube, hallelujah, transformation occurs. In order for this transformation to succeed, the E. coli bacteria must be competent, which means ready to accept the foreign DNA from the environment. This competence is ensured by treating the cells with calcium or magnesium. Don't worry too much about how this competence business really works. Just know that bacteria must be competent for transformation to occur.
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