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Name: Jenny W.
Status: Student
Age: 17
Location: N/A
Country: N/A
Date: N/A 


Question:
I go to a high school that is relatively untechnologically advanced, but have access to the equipment in the forensics lab of a police department about 45 minutes away from where i live. For an independent research project I caused a reverse mutation of the lacY and lacZ genes in two E. coli strains that had been lactose intollerant. The mutation caused them to be able to ferment lactose. I would like to further this experiment, but I am at a loss as to what my next step should be.

What does recombinant DNA involve? What equipment is needed to do it and how do you know what genes a bacterium will accept and what ones will kill it? What DNA is the best to try and introduce?



Replies:
Your first question: what does recombinant DNA involve? This term is used for experiments where DNA is artificially altered, and then put back in an organism, in your case an E. coli.

There is not much equipment involved when you do the transformation with competent E. coli cells. These you can make yourself but you would need a centrifuge for that, a waterbath, and the usual bacteriological equipment that you obviously have (petri dishes, incubator, etc.). You would need vector DNA which can be purchased. Maybe a nearby microbiology department could donate some.

The simplest experiment is to introduce a resistance to an antibiotic. Antibiotics are compounds that kill bacteria, but bacteria can become resistant to it. They do this in two ways, either they mutate spontaneously their own DNA in a special gene that makes them resistant, or they take up DNA (this is called transformation) that helps them become resistant. You could do the following experiment: compare the efficiency to become resistant to an antibiotic by spontaneous mutation, versus transformation. Check the exibit on 'antibiotics' in the Virtual Museum of Bacteria

(www.bacteriamuseum.org/niches/hwfbacteria/antibiotics.shtml)

to do a bit of reading first. Ask your supervisor which type of antibiotic, and antibiotic-resistance gene (present on a plasmid) is available to you.

Another experiment you could do is to see which mutation is easier to introduce in E. coli, resistance to the antibiotic or tolerance for lactose. You would need to do some calculations to determine the frequency of mutation.

Other suggestions for experiments you can also find in the museum, see

www.bacteriamuseum.org/educational.shtml

Have fun with your bacteriology experiments!

Trudy Wassenaar
Curator of the Virtual Museum of Bacteria



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