Week 6

In Dr. Kirkman’s lab, I learned and have been applying an interesting technique known as TOPO for cloning amplified DNA from the beta subunits of the p. falciparum proteasome.  TOPO is a PCR cloning technique that involves inserting PCR amplified DNA products into plasmid vectors for cloning.  The technique is based on the functionality of topoisomerase I, which is an enzyme purified from the vaccinia virus and is capable of catalyzing breakage and re-ligation of phosphodiester bonds in DNA.  The functionality of this enzyme is exploited during the TOPO procedure to enable insertion of blunt end DNA into the plasmid cloning vector.  Following insertion, competent E. coli cells are heat shocked to facilitate uptake of the plasmid, which also contains a kanamycin antibiotic resistance gene.  The propagation of E. coli on LB agar plates with kanamycin and subsequent inoculation of single colonies in liquid LB media with kanamycin is quite conducive to the growth of cells that successfully undergo plasmid uptake.  Plasmid DNA can be extracted from E. coli cells in the liquid culture through the alkaline lysis based miniprep procedure.  Restriction digest and gel electrophoresis are then used to assess the efficacy of TOPO cloning by verifying the size of the cloned inserted DNA fragment.  An added benefit of using TOPO plasmid vectors for cloning is the ability to use the universal priming sites (e.g. M13, T7) contained within the vector for accurate sequencing.  This is especially useful when working with the p. falciparum genome, which can be extremely AT rich in certain regions which poses a challenge for primer design.  Over the last two weeks, I have been able to TOPO clone DNA from four of the seven beta subunits.  Hopefully, I’ll be able to finish the rest of the beta subunits by the end of next week.

In the clinic, I shadowed Dr. Vielemeyer during inpatient service as well as his teaching rounds with the new infectious disease fellows.  This was a great learning experience since I had the opportunity to observe the advanced infrastructure of the hospital’s intensive care units and also learn about the expectations placed on medical fellows in training.