Which antibiotic resistance gene does my plasmid have?

February 8, 2011 at 2:28 pm | | cool results, everyday science

Few experiments in science are conclusive.  So, it is very exciting when an experiment provides completely unambiguous results.  Today that happened.

Molecular biologists use bacteria–specifically strains of E. coli that don’t make people sick (i.e., non-pathogenic)–to make lots of copies of circular DNA, called plasmids; one can think of E. coli as a photocopier for plasmids.  Bacteria love to take up plasmids, copy them, and express them to make proteins with unique functions, and it is this ability that makes them so evolutionarily successful.  Bacteria can transfer plasmids containing antibiotic resistance genes to each other, for example, and in this manner, can become “superbugs.”  Staph is one pathogen that has done this so successfully in hospitals that we will soon run out of antibiotics to treat it.

Interestingly, molecular biologists give E. coli antibiotic resistance genes on purpose. It’s not because we’re bioterrorists.  Rather, we want to be able to SELECT for those bacteria that take up our plasmid of interest and copy it.  So we make sure that the plasmid DNA that we give the E. coli to copy also has a gene that codes for a protein that confers antibiotic resistance; ampicillin resistance is the most common.  We take this plasmid, mix it with the E. coli, and warm the E. coli slightly.  This creates little pores in the E. coli that allow the plasmid DNA to pass through.  The process–called heat transformation–is not very efficient, and most of the E. coli don’t take up any plasmids.  We don’t want to grow these E. coli because they are useless to us; we only want to grow the ones that took up our plasmid.  So, we add some ampicillin, and only those E. coli that took up our plasmid DNA can survive.  As a result, we end up with a bacterial culture that is loaded with our “photocopied” DNA.  The resulting plasmid DNA can be used for many things; for example, DNA that codes for insulin can be copied in E. coli, purified, and then put into mammalian cells to cause them to make insulin, which can be harvested and given to diabetic patients.

I recently wanted to use E. coli to “photocopy” some plasmid DNA that I got from another researcher, but the researcher wasn’t sure which antibiotic resistance gene for selection was employed in the plasmid (not good record keeping).  He thought it was either ampicillin or kanamycin.  So, I transformed E. coli with the plasmid and tried growing the bacteria in both ampicillin and kanamycin.  Take a look at the picture below.  It’s pretty obvious that the bacteria survived only in the kanamycin (yellow cloudy suspension on the right), indicating that the plasmid coded for kanamycin resistance.  The bacteria in the ampicillin died quickly and did not grow.

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  1. hey nick! thanks for the post. i’ve been neglecting the blog for a while, eh?

    Comment by sam — February 8, 2011 #

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