ISS C. elegans Experiment Questions

Model Organism: Worms Scientific Discussion
1

Hello all,

An experiment I proposed has been tentatively selected to go aboard the International Space Station. I was notified of this a couple of days ago and was caught off guard by the schedule, so even though I know I should perform experiments to find out the answers, I was hoping that some of my questions could be answered on this forum. I still plan to perform the experiments myself and am in the process of obtaining all of the materials.

  • I plan to sustain culture growth inside two 75 mm long polypropylene plastic tubes with 12 mm diameters. I will fill each tube with 5 mL of LB Agar. The leftover space will contain oxygen. Will this be enough broth and/or oxygen to sustain culture growth for approximately a month and a half? What is the carrying capacity of LB Agar broth?
  • What would be an ideal initial culture size?
  • What is the approximate population doubling time of wild-type and daf-2 modified C. elegans?
  • If the growth rate of the cultures will deplete the broth too quickly, will growth slow to the L1 or the dauer stage for a long enough period of time for the experiment to last until I receive the results for examination?

One worry I have about my current experimental design is whether or not the C. elegans will saturate the carrying capacity of the broth. Another option would be to use starved larvae and have the astronaut kick start growth in space by breaking glass vials and thereby mixing starved larvae and broth. Would this option be more viable?

I would be very grateful for any information you could share with me. Thank you for your time!

2

I would encourage you to review the details of worm growth described in the WormBook (http://wormbook.org/chapters/www_strainmaintain/strainmaintain.pdf). You’ll discover that worms aren’t propagated on LB agar, that growth rate and generation time are influenced by temperature, etc. I assume you plan to assay lifespan, given the inclusion of the daf-2 strain; you should review some of the relevant literature, too.

-Harold

3

Also, LB agar wouldn’t have a broth, it would be solid. If you plan on growing them in liquid culture, I would HIGHLY recommend you make sure you have that working in your hands beforehand, as it can be tricky to get to work.

4

Sorry, meant to include OP50 pellets in my experimental design description. I had LB Agar confused with S medium.

Time constraints mean that, whatever my design, I will receive dauer-stage C. elegans. I will aim, therefore, to examine the effects of microgravity and increased levels of radiation on cells, structures, systems, lifespans, and DNA of dauer-stage larvae of daf-2 modified C. elegans compared to a dauer larvae of a wild-type strain also grown in space and dauer larvae of two cultures of the same strains also grown on Earth.

After reviewing WormBook and C. elegans: A Practical Approach, I have decided to use S medium as the liquid media in question. Beginning in January, I will have a month and a half to perform optimization assays at a local lab on broth make-up, OP50 pellet concentration, and lifespans. I must, however, submit a list of materials I plan to use by Thursday morning, before I have the chance to begin assays. S medium, OP50 pellets, starved larvae of both wild-type and daf-2 modified are the materials I have listed, including the items I need to create the medium and the pellets (L broth, S basal, trace metal solution). Do I need anything else to grow C. elegans in a liquid environment?

The lab should be equipped with the necessary machines (autoclave, centrifuge, automated shaker, multi-channel micro-pipette, incubator, freezer). I hope to obtain the strains from the CGC.

As a high school student, it is difficult to know where to begin reading relevant literature on this subject. I am in the process of reading several articles related to the daf-2 modification, previous C. elegans experiments in space, and C. elegans: A Practical Approach. What are some other “basic” books or articles I should read to familiarize myself with this topic?

5

A few questions that you should take into consideration:

What temperature are these experiments going to be performed at?
What is your sample size?
How will you monitor deaths?
Which allele of daf-2 are you going to be using?

Instead of only having 2 vials, can you perhaps have 4? It’s always good to have a backup lifespan experiment running at the same time incase something happens to your experimental sample.

Although not all of this JoVE pub is relevant to you, it’s worth checking out: Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates | Protocol

Just a general note: I’m not sure if you’ll have enough time (1.5mo) to complete trial runs for your lifespan assays.

6

Based on correspondence and articles I’ve read:

Temperature: 22 degrees C ± 1 (ambient temperature aboard ISS), unless I use an incubator (TBD)

Sample Size: I’ll perform assays to determine. I’ve chosen to activate my experiment 21 days before it leaves the ISS. Given past experimental designs, this should be more than enough time to grow a sizeable amount of worms. I may be able to use C. elegans Maintenance Medium (CeMM) in my experiment, which would eliminate the need for OP50 and reduce the drain on oxygen. I just need to find the approximate growth rate of C. elegans and how long it takes to get to my target number, which I’ll determine once I hear back from some Professors who are willing to work with me analyzing my results.

I won’t be able to measure deaths actively aboard the ISS. Here are the things I’m planning on looking at, with help from knowledgeable persons: genomic and proteomic changes (both in muscle systems and in metabolic systems such as Insulin-like and TBF-beta-like signaling) and lifespan and broodsize. I’ll use the wild-type as a control (to determine validity of results, I’ll compare with data sets from past experiments). So, I’m looking as possible atrophy, possible changes in the lifespan of daf mutants, and possible changes in metabolic functions.

I am familiarizing myself with daf-16 and daf-2 before I make a final choice, but as of now it’s looking like daf-16 would be more temperature resistant (which would help during transit). That, combined with the fact that daf-16 is supposed to be responsible for some of the mutations previously noted, makes me lean toward daf-16, but I’m still reading up on the subject.

I can’t have 4 vials - this is run as an educational opportunity, not a research opportunity, and as such I’m limited.

The link is great, thank you for the video!

7

Sample size is very important and you’ll need to ensure that your sample size is consistent with your controls. I’m still confused with your lifespan assay. Are you saying that you want to only have 2 data points for your Kaplan-Meier curve (ie. 100% alive —> x % alive at the end of your experiment)? For your other measurements regarding metabolic function, how are you planning to measure these? Will these be performed on the ISS or back on Earth?

For your assays to determine genomic/proteomic changes, I feel that you’ll need a rather large population (thus my concerns regarding sample size) but I don’t know what kind of experiments you’re planning on doing.

8

On Earth, the daf mutation extends lifespan. I can’t measure the lifespan of creatures who grow and die during the course of my experiment (not possible with the hardware I’m using), but I am interested in knowing the effect of low earth orbit on the lifespan/development of the daf mutants. When my organisms return from the ISS, I will isolate a population of L1 or dauer-stage larvae and restart growth. I begin my Kaplan-Meier curve then and compare that curve to a curve I’ll take of the control group that was grown on Earth. Essentially, I’ll be measuring the effect of LEO over several generations of growth on the extended lifespan phenotype (compared to a control on Earth, of course). I’m curious as to what the effect of several generations of growth will be on the daf mutant extended lifespan phenotype. Previously, LEO growth was shown to partially rescue a paramyosin mutant. Although that’s a muscle mutant, it sparked my interested in seeing what LEO will do to a metabolism mutant. Since then, I’ve learned that LEO has also been shown to change the expression of many daf-regulated genes.

I’m not quite sure what experiments I will do to for my metabolic/proteomic/genomic analysis. I’m going to meet with several scientists at a local Worm Lab to try and figure that one out. Most of my experimental design is set, now - all I need to do is work with these scientists on performing actual assays and gaining the knowledge and experience necessary for the data analysis!