Week 2: Into the Lab!


Lindsay Holsen is our CEC/RTC summer intern, visiting us from Lawrence University in Appleton, WI. In addition to community engagement and research translation work, Lindsay is helping doctoral trainee Tess Leuthner with a fascinating research project on soil nematodes and contaminated soil. 

Research is like baking.  You get your recipes down in one oven and then you go over to grandma’s house and the oven just doesn’t work the same!  In the lab you get comfortable with your protocols (a recipe for lab techniques), but even if you follow every step at a painstaking pace, exactly the same, there are always those inexplicable differences and challenges.   

However, I tried to circumvent the “different oven” with the “same recipe” issue in my summer transition from Lawrence University to Duke University by choosing a project that will force me to learn completely new techniques.   I am fortunate that Tess Leuthner, a doctoral trainee at the Nicholas School of the Environment, kindly took me on as an assistant this summer to work on her side project that utilizes Caenorhabditis elegans to detect heavy metals in community garden soil samples.  

MeetC.elegans!  

Not only just your friendly neighborhood soil nematode, these roundworms have incredible utility in biomedical and environmental research from oncology to toxicology and so much more!   

Image: WormAtlas. Altun, Z. F. and Hall, D. H. (ed.s). 2002-2006. http://www.wormatlas.org 

 

But why are C. elegans so useful? 

There a many factors that make them applicable and useful in many contexts: 

  1. They can be maintained at a low cost 
  1. Their activity/responses can be shown in a  whole, living animal with working digestive, reproductive, endocrine, sensory and neuromuscular systems rather than a simple cell culture (Hunt, 2017). 
  1. They have been used in comparisons of toxicity across different species, including rats and mice, which can tell us more about the impacts in larger organisms such as humans (Hunt, 2017) 
  1. Their short, well understood life-cycle, resilience to stress, and well characterized genome (Leung et al, 2008) help researchers notice differences and stress responses to different toxicological changes in the environment 

These abundant worms bring a lot to the table, so how do they help us understand if heavy metals are in the soil?   

Their genome has been fully characterized, which provides the opportunity for researchers to selectively add special reporter genes to tell us specific information about the nematode’s responses to environmental conditions.  Those reporter genes, like green fluorescent protein (GFP) for example, are paired with a specific gene in the worm that then triggers the green glow of the GFP when its “partner gene” is activated in the presence of an environmental trigger.  In the case of the special strain of C.eleganswe’ve chosen, it utilizes that same reporter, GFP, to glow green when heavy metals are present because it is coupled with metallothionein (MT).  Metallothionein is a gene in C.elegans (and humans too) that is activated as a stress response to heavy metals, making very useful in our quest to detect them in community garden soils!    

The squiggle you see above is my simplified C.elegans.  The diagram shows the pairing of the reporter gene, GFP, with Metallothionein-II (mtl-2) which is activated by the environmental presence of heavy metals near the worm causing the green glow!  The fluorescent microscope will be a great tool for visualizing that fluorescence and comparing the fluorescence levels we find for different community garden soil samples across the state of North Carolina! 

We’ve collected a few samples so far, but it will definitely take a few more steps before we can compare the C.elegans stress responses to heavy metals in the soil samples from different gardens …  

So what are we working on right now? 

Chunking and Picking…. essentially feeding the worms! 

Everybody needs to eat and the choice meal of C.elegans is not exactly lasagna.  They eat Escherichia coli (abbreviated E.coli)!  They reproduce so quickly that they need to be fed about every 2 days because they eat up all the E.coli on the plate.  However, there a few ways to feed them and still maintain reasonable population sizes. 

a. Chunking:  Cut out a piece of the growth medium, agar (about 1cm2), from the plate with the worms currently on it and flip it over lightly, placing it face down onto a fresh plate of E.coli. Voila!  Let them feast! 

b. Picking:  is especially helpful for making sure the population doesn’t grow too fast, especially during a period when you can’t give them quite as much attention for a few days.   A metal “pick” embedded into the melted tip of a glass pipette, gets dabbed gently on the edge of a fresh plate of E.coli and is then lightly tapped onto individual C.elegans to get them to stick to the tip for transfer to the fresh plate of E.coli.  A microscope is especially helpful for ensuring a successful transfer! 

For each of these techniques, the metal tools used are flamed with ethanol to avoid contamination (and we also try to avoid contaminated parts of the plate).  This, in addition to regular feeding, helps avoid putting any extra stress on the nematodes before the real test: HEAVY METAL!! 

What are our upcoming goals? 

From here we… 

  1. Hope to test the best way of preparing the collected soil samples so we don’t introduce extra variables
  2. Test an exposure of the worms to the soil
  3. Designate a “control” soil sample with safe levels of heavy metals
  4. Expose the worms to the soil samples, visualize, and compare!

 It’s not quite a brownie recipe, but we’ve got some great things cooking this summer!  Keep an eye out for future updates about our adventures with soil and nematodes! 

Behold, the fluorescent C.elegans

 

 

References:

Barrière, A., & Félix, M. A. (2006). Isolation of C. elegans and related nematodes. WormBook, 17, 1-9. 

Hunt, P. R. (2017). The C. elegans model in toxicity testing. Journal of Applied Toxicology, 37(1), 50–59. http://doi.org/10.1002/jat.3357 

Leung, M. C. K., Williams, P. L., Benedetto, A., Au, C., Helmcke, K. J., Aschner, M., & Meyer, J. N. (2008). Caenorhabditis elegans: An Emerging Model in Biomedical and Environmental Toxicology. Toxicological Sciences, 106(1), 5–28. http://doi.org/10.1093/toxsci/kfn121