Dicty and the DNA Drama

This summer, I don’t even miss my shorts and flip-flops.  I prefer the lab coat, actually.

Each phase of the fruiting body formation is shown.

Dicty Fruiting Body Development

I have willingly traded summer essentials for lab gear and the opportunity to work with Dr. Parrish in her Dicty lab.  We have spent the past three and a half weeks working on Dictyostelium discoideum.  This social amoeba is fascinating, as it is unicellular when properly fed, but cells will aggregate and differentiate to form a multicellular fruiting body which will release spores once conditions improve.  For my particular project, I am focused on a gene similar to the L534 gene in Mimivirus.  This L534 gene is responsible for coding a mRNA decapping enzyme.  My interest is in characterizing this similar gene in Dicty by observing the phenotype of Dicty without this gene.

Cell cultures are displayed on a device used for counting the cell concentration.

AX3** cell culture on left and L534 KO cell culture on right, on a haemocytometer for cell counting. (Click for a larger image.)

Previous research students have created the Dicty L534* knockout, and my task is to study the impact of the L534 gene’s absence to develop a better understanding of the gene’s function and importance in Dicty’s development and survival.  There are three stages of my research which will provide details that we are seeking about the L534 gene in Dicty:

  1. Confirming the Knockout
  2. Observing Growth Rate
  3. Observing Cell Differentiation

The Dicty cultures have finally become healthy enough for completing these phases of the L534 knockout characterization, however, we have reached a snag in our master plan.  To confirm the success of the knockout, we must extract genomic DNA from the Dicty and amplify it though PCR as well as visualize it through gel electrophoresis.  We have completed the extraction and PCR, and we have run the gel, but much to our dismay, the DNA seem to have been degraded.  Confirming the knockout is imperative to the continuation of the L534 characterization, as without the proper knockout of L534, we could not study the effects of the gene.  And so, we have reached a determinant point.  We will be repeating the genomic DNA extraction, PCR, and gel, and the outcome will either “make it or break it.” Stay tuned for the resolution to our obstacle…

*For convenience, we call the gene of interest in Dicty “L534,” though this is the name of the similar gene in Mimivirus rather than the actual name of the gene in Dicty. **The AX3 Dicty cells will serve as the wild type as a means of comparison.  The AX3 cell culture is a strain of Dicty that can be grown axenically, meaning that it is grown on a nutritional substitute (HL5 medium) rather than the bacteria on which it normally feeds.