A Study in Soil

Episode number: Season one, episode one

Directed by: Dr. Jacobs and Dr. McMillan

Written by: Xi Huang

Starring: Invertebrates

Mary Chow

Shirley Mancia

Xi Huang

Dr. Molly Jacobs a.k.a. Molly

Dr. Brett McMillan

Air date: June 9, 2013

From left to right. Xi Huang, Mary Chow, and Shirley Mancia.

Getting soil samples.

Getting soil samples.

“A Study in Soil” is the first episode of the TV series The League of the Green Hornet and first broadcast on June 9, 2013. The series started its production on May 29, 2013. It took three days of careful planning and getting ready. List of supplies and specific plans were made in order for filming to happen. On June 6, the team finally started shooting under the supervision of Molly.

 

PLOT

During the summer of 2013, in an ordinary neighborhood called Hashawha, three private investigators (Mary Chow, Shirley Mancia, and Xi Huang) joined forces to solve a mysterious case: whether trails in Hashawha act as a barrier in the migration of soil invertebrate communities.

A total of 60 soil samples will be collected from eight different sites. Nine samples will be from each of the four path sites: three samples from the center of the trail, three 3 m from the creek, and three 8 m from the creek. Six samples will be collected from each of the four non-path sites: three 3 m from the creek and three 8 m from the creek. The purpose of doing this is to test if soil invertebrate communities change on a gradient away from stream.

Detective Shirley Mancia recovers her thermometer.

Detective Shirley Mancia recovers her thermometer.

While the investigators were working on their first site, something unexpected happened. Detective Shirley Mancia lost her precious thermometer! After Inspector Mary Chow poked a 9-inch-deep hole in the ground with a Soil Compaction Tester, Detective Shirley Mancia accidentally dropped the thermometer that was used to measure soil temperature down the 9-inch hole. In a moment of panic, Shirley started digging frantically. Fortunately, the thermometer was recovered. So once again, the day is saved. thanks to the League of the Green Hornet. But will they be so lucky next time? Until next episode.

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.


 

 

 

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!