Lord of the Dicty

Hello everyone! I cannot believe that Friday was our last day in the lab! It has been such a great learning experience. In this time I have successfully extracted Dictyostelium genomic DNA and amplified the two flanking genomic regions surrounding the Dicty DDB_G0278957 gene, which encodes a putative mRNA decapping enzyme. I then cloned the flanking regions into a bacterial plasmid and introduced the plasmid to bacteria.

 

This picture shows the gel resulting from our restriction digest. This digest demonstrates that the bacterial plasmid DNA contains our insert. It was very exciting to see that all our work paid off.

We then used this plasmid to knock-out the Dictyostelium DDB_G0278957 gene by homologous recombination. The knock-out plasmid was engineered to confer blasticidin resistance and we were able to obtain D. discoideum colonies that were blasticidin resistant, suggesting that we were successfulin knocking out the DDB_G0278957 gene!. The next step is to obtain clonal isolates of the knock-out strain by diluting the cells and growing them on bacteria. The entire process of a Dicty transformation can take over a month! Once we confirm that the DDB_G0278957 is knocked out, we will analyze the biochemical consequences of the absence of the DDB_G0278957 gene and protein.

One of the coolest things we have done was to look at developing D. discoideum cells. When starved for nutrients, Dicty cells aggregate together to form a multicellular fruiting body containing spores that will be released when conditions are more favorable. The process of development takes 24 hours and we were able to induce development and see six developmental stages. We harvested these different stages to isolate mRNA and examine the expression pattern of DDB_G0278957 during development by Northern blotting. We started work early and plated the cells on non-nutritive filters. Throughout the day (and night), we looked at the plates under a dissecting microscope and observed the different developmental stages.. The first stage we observed was ripples. The cells gave the filters a shiny brown color and only four hours into development, waves could be seen forming on the plate. Four hours after that we saw loose mounds forming. By 11:30 at night these mounds had grown tips. It was really cool to see the cells changing. The next morning the cells had formed the final fruiting body stage containing the spores. Unfortunately, we did not have a camera set up to take pictures of the different stages as seen from a microscope but I have found this picture for you:

By Tijmen Stam, IIVQ (SVG conversion) – user:Hideshi (original version) (en:Image:Dicty Life Cycle H01.png) [GFDL (http://www.gnu.org/copyleft/fdl.html), CC-BY-SA-3.0 (http://creativecommons.org/licenses/by-sa/3.0/) or CC-BY-SA-2.5 (http://creativecommons.org/licenses/by-sa/2.5)], via Wikimedia Commons

Below you can see Dicty growing on plates containing bacteria. The clear regions are the regions where the Dicty cells have eaten the bacteria. As they deplete the bacteria, the cells aggregate and undergo development. The “fuzz” seen on the plates are the fruiting body containing spores.

This summer I have learned so much about molecular biology and have had a great time working with Kirsten and Dr. Parrish. Thank you Dr. Parrish and McDaniel for giving me such a great opportunity!

Driving Miss Dicty

We are currently in our fourth week of research here at the McDaniel Dicty Lab. Dr. Parrish, Kirsten Bickford, and I (Catherine O’Keeffe) are studying the social amoeba Dictyostelium discoideum, also known as a slime mold. We are interested in looking at two particular genes that code for possible mRNA decapping enzymes.  We started this project at the very beginning, which means had had to grow the amoeba cells from stocks. Since our first days in late May we have been growing and passing cells. Luckily, the cells have been healthy and we only had one unfortunate case of fungal contamination. We have been mastering many molecular techniques such as DNA extraction, PCR, gel electrophoresis, ligation, and transformation.

One thing that surprised me about working here is the amount of independence we have. Before this summer, I was not too confident in my lab skills. However, now I am much more self-assured and feel motivated to work on my own initiative. One of my favorite things to do is troubleshoot (even though this means that something went wrong). Though we are just undergraduate students, Dr. Parrish asks us what we think needs to be changed and has us research to figure out how to fix the experiment.  We look at the protocols, search online, and talk together to figure out a solution.

Working in the lab is also a lot of fun! We listen to music, have glar dates together, and joke around. Enjoy these pictures: