Summer 2013!

It’s finally summer 2013, and this year the Jacobs lab is working locally, in and around McDaniel’s campus.  We’ve teamed up with botanist/ecologist Brett McMillan, and we will be studying the mysterious world of soil invertebrates (more on this soon).

We’ll be joined on the blog this summer by the Becker lab (Environmental Science), and the McKenzie/McCole lab (Exercise Science & Physiology).  Stay tuned!

One Hundred and One (Million) Dicty

Hello everyone! Last week was our final week of research on Dicty and it was most definitely bittersweet. Although Catherine and I are excited to have the rest of our summer, it seems that we may have successfully created transformants from our gene knockout project! We replaced the Dicty Nudix gene DDB_G0283315 (also known as DCP2) with a blasticidin resistance gene, which was a long but surprisingly easy process. Through the polymerase chain reaction (PCR) we amplified the right and left flanks around the DCP2 gene and cloned them into a bacterial plasmid surrounding the blasticidin resistance gene.  The flanking regions of the DCP2 gene should promote homologous recombination in Dicty, allowing the DCP2 gene to be replaced with the blasticidin resistance gene. Blasticidin is an antibiotic lethal to many prokaryotes and eukaryotes and only Dicty cells that have the blasticidin gene in place of the gene to be knocked out should grow.

To insert this plasmid into our Dicty cells, we linearized the plasmid and then used an electroporator to shock the DNA into the cells. We then added these cells to media without blasticidin for a few days, and once they seemed to be growing again we started to add blasticidin to select for the Dicty DCP2 knockout. At first the results were not promising, because even our control plates seemed to be growing the same as the knockout plates. After about two weeks, there was a marked difference between the controls and the transformants. When Dicty is healthy, it will adhere to the bottom of a petri dish in liquid media. The control cells were most definitely dead because they were all floating and very small and round. The transformed cells from the DCP2 knockout had many floating and clumped cells yet areas of healthy cells growing as colonies. When we found these colonies, we were all very surprised! The presence of these colonies confirmed that blasticidin resistance had been conferred into our Dicty cells! None of us expected success in this experiment because the gene could be essential for the cells to survive and/or Dicty transformation is a notoriously difficult and long process. Needless to say, we are thrilled with our results, but much work still needs to be done to confirm our gene knockout.

We next wanted to isolate clonal populations of Dicty that had the DCP2 gene knocked out.  To do this, we diluted the transformed cells and started to grow them on LP agar plates with B/r, a strain of E. coli bacteria. Dictyostelium eats bacteria in its natural habitat, the forest floor, so once our Dicty cells begin to consume the bacteria Dicty plaques will form on the plates.  A plaque is a hole in the bacteria that is assumed to have been initiated by one Dicty cell, thereby producing a clonal knockout population. When the cells have eaten all of the available bacteria in the area, fruiting bodies will form which contain spores. Then we will isolate the sporeheads and begin growing them in regular media with blasticidin.  Next, we will need to confirm the gene knockout by either PCR or Northern Blot. There is a possibility that the blasticidin resistance gene was put somewhere else into the genome rather than where the DCP2 gene is located, so while our cells seem to be blasticidin resistant, we cannot be completely certain of the knockout until further screening is done. Unfortunately I will not have time to screen these cells further, so Dr. Parrish will generously be coming in for the rest of the summer to finish the experiment. We all hope that the knockout is confirmed, but then we need to assess the consequence of the loss of this gene for Dicty cells.   We will look at the effect on mRNA turnover in these knockout cells, since DCP2 encodes a putative mRNA decapping enzyme.

 

In the last week of research we performed a Northern Blot, which is a method to measure levels of gene expression during different times of development. As you learned in Catherine’s post, we performed a time course last week and harvested the cells at different stages in their development. After extracting RNA from the cells we ran a gel to resolve the RNA. The blotting part of the experiment involved placing a membrane under the gel and a weight so that the RNA was transferred onto the membrane. After this was finished, we treated the membrane with luminescent probes for the gene of interest and visualized the results by film development with autoradiography paper.

  

Unfortunately, our film did not turn out as desired but this is most likely due to a problem with the blotting procedure. There was a lot of background on the X-ray film, making the results hard to interpret. There may have been problems with the actual transfer of the RNA to the membrane or possible RNA degradation, yet in either instance the experiment must be repeated. Dr. Parrish and future research students will troubleshoot the experiment to receive better results.

Summer research was an invaluable experience that I will remember for the rest of my life. Although Catherine and I worked hard (most of the time), we had a ton of fun in the lab and kindled a new friendship. Dr. Parrish was wonderful with helping us get a better grasp of our projects and a better understanding of molecular biology in general. We hope that you enjoyed our posts and enjoyed learning more about Dictyostelium!

~Kirsten

The Fellowship of the Dicty

Hi again!

Last Friday was the last day for Kirsten and Catherine in the Dicty lab at McDaniel College. Although the summer was extremely busy for all three of us, I was still extremely sad to say goodbye to these bright and talented young ladies. As I sit in my office today, I miss hearing their giggles from my lab next-door, listening to their entertaining stories, and watching them grow as young scientists.  I think this means that I am in the right profession!

I referred to Catherine and Kirsten as my “miracle workers” this summer.  They were extremely successful with their experiments and accomplished more than I even anticipated.  I will let them tell you about their exciting results in their own blog posts coming soon.  However, to understand how much work they accomplished, here is a sampling of some of the techniques they mastered this summer:  Dicty cell propagation (both on media and bacteria), Dicty development, Dicty genomic DNA extraction, the polymerase chain reaction, agarose gel electrophoresis, restriction enzyme digestion, ligation, bacterial transformation and selection, plasmid purification, Dicty transformation and selection, Dicty RNA extraction, Northern blotting, and autoradiography.  Perhaps most impressive of all, they learned how to do science, including how to plan experiments, perform the proper controls, and interpret their results.  They plan on presenting their work at a conference in the fall and this research will also be the focus of their senior capstone poster and paper next spring.

I want to leave by saying that everyone always says that the young have much to learn from the “old”.  While this is certainly true, I continue to learn so much from my students.  They remind me not to take myself so seriously and that humor can be found in the every day experience; I just have to change my frame of reference.  For example, while exiting the darkroom, the three of us became trapped in the dark room revolving door, basically a small enclosed tube that is in complete darkness.  After several harrowing minutes of being trapped, we finally managed to get out of the door.  My students stayed much more calm than me, and our perilous situation became the running joke of the week because of their light-hearted way of seeing the situation.  The door, post-incident:

Another example of their refreshing attitude related to our experience with a temperamental autoclave (basically a big pressure cooker) that we use to sterilize media and equipment.  On the last day, Kirsten and Catherine gave me a hot glove for taking hot items out of the autoclave.  On it they had written “ Autoclave Survivors 2012” “Living in Peril to Keep Things Sterile”.  (Don’t worry; we weren’t really in danger from the autoclave!)

I also received as gift a CD that had a picture of our shaking incubator as the cover.  All of the songs on the CD have the word “Shake” in the lyrics.   The gift bag also had a picture of Catherine and Kirsten embracing the autoclave.  How did I get so lucky to have such creative, kind, and bright students? :)

Our final day and a visit to “The Cow” for a frozen treat.


 

 

 

Field Notes from Madagascar

Here’s a note from Dr. Morrison, currently doing field work in Madagascar.  His location is extremely remote with no internet access, but he was able to send us this note & photo during a brief trip to town! “I am back for the night to Majunga from our field site in Madagascar. I am having a fantastic time and finding lots of chameleons and leaf-tail geckos. Last night I saw in the field and caught my first Uroplatus henklei, just the third of the season this year. These are truly stunning animals. Student projects and survey routes are going well. Back to the field in the morning”

Dicty Dancing

Hello everyone! My name is Kirsten Bickford and I am doing summer research with Catherine O’Keeffe and Dr. Parrish. We are using the amoeba Dictyostelium discoideum to eventually isolate putative mRNA decapping enzymes. These enzymes, termed Nudix enzymes, are found in large viruses all the way up to humans so being able to isolate and define the function of these enzymes is very exciting.

In order to do our experiments, we need a constant stock of healthy cells. We grow the Dicty cells on plates and in a flask and usually pass them about 3 times a week, so as to diminish genomic mutations. So far we have isolated genomic DNA, and are starting the process of doing a gene knockout. Currently, we are ligating and transforming parts of the gene into bacterial cells, but this is just the beginning of the process.

Compared to working in class laboratory settings, summer research is much more independent. Although I have learned sterile technique previously, there is much more pressure to do it properly as our cells will become contaminated otherwise. Many of the molecular techniques are new to me but with Dr. Parrish’s supervision, I have been able to learn how to do them properly and on my own. Pipetting is a huge factor and my technique has definitely improved since the start of the summer.

Hopefully our research will continue to go as planned, without equipment failures or major contamination. We will definitely be busy for the next 5 weeks, but another blog post is coming soon!