In this experiment we investigate the effect of a 30mM concentration of caffeine on transgenic CL-2070 C. elegans. A cell under stress will upregulate proteins known as heat shock proteins (HSPs) that function as chaperones to refold misfolded proteins due to stress. Caffeine, a chemical stressor, has been shown to induce HSP response, alter movement and feeding behaviors, disrupt early larval development, and shorten the lifespan of C. elegans (1). Caffeine may induce HSP16.2 transcription and translation by binding to the HSF-1 transcription factor, thereby increasing its affinity for the heat shock element within the promoter.

1. Induction of HSP16.2 Response in C. elegans Using Caffeine as Stressor
By Kennedy Jackson, Brittany Perryman, Rita Stevens, and Kali Wageman
The Evergreen State College
Introduction
In this experiment we investigate the effect of a 30mM
concentration of caffeine on transgenic CL-2070 C. elegans. A cell
under stress will upregulate proteins known as heat shock
proteins (HSPs) that function as chaperones to refold misfolded
proteins due to stress. Caffeine, a chemical stressor, has been
shown to induce HSP response, alter movement and feeding
behaviors, disrupt early larval development, and shorten the
lifespan of C. elegans (1). Caffeine may induce HSP16.2
transcription and translation by binding to the HSF-1
transcription factor, thereby increasing its affinity for the heat
shock element within the promoter.
Hypothesis
We hypothesize that caffeine will induce HSP stress response,
prevent early larval development, and shorten the lifespan of
transgenic CL2070 C. elegans.
Results
Worms:
Populations were scarce (1-6 worms per plate)
Generally small in size
Slow moving
Analysis:
GFP expression was low
SDS-PAGE showed bands in all lanes (RNAi, no RNAi,
and positive control)
Western blot showed bands for positive control only
Methods
Discussion
The combined results indicate three possibilities:
1) Not enough worms survived the initial stress
2) Length of lifespan and development were affected
drastically
3) Caffeine stress did not induce HSP16.2 stress response.
Due to the absence of a control group for caffeine treatment,
we cannot accurately determine the effects of caffeine
treatment. We can claim that RNAi was successful with little
confidence, as chi-square analysis with low sample population
is unreliable.
We reject our hypothesis at this time. A repeat experiment
might use a lower concentration of caffeine to avoid
termination, and incorporate a control group with which to
compare stressed worms.
Literature Cited
1. Al-Amin, M., Kawasaki, I., Gong, J., & Shim, Y.-H. (2016). Caffeine Induces the Stress Response and Up-Regulates Heat Shock Proteins in Caenorhabditis elegans. Molecules and Cells, 39(2), 163–168. http://doi.org/10.14348/molcells.2016.229
2. Echeverri, D., Montes, F.R., Cabrera, M., Galán, A., & Prieto, A. (2010). Caffeine's Vascular Mechanisms of Action. International Journal of Vascular Medicine, vol. 2010, Article ID 834060, 10 pages. doi:10.1155/2010/834060
3. Smatresk, N. J. (2002). How does caffeine affect the body? Retrieved February 13, 2017, from https://www.scientificamerican.com/article/how-does-caffeine-affect/
5’-nGAAn-3’
HSF-1
Figure 1 C. elegans cell. When cells are stressed, the
transcription factor HSF-1 binds to a heat shock element with
a sequence of 5′-nGAAn- 3’ within the hsp16.2 promoter,
resulting in transcription and translation of the heat shock
protein, HSP16.2.
Stress
HSP16.2
No glow
Glow
Figure 2 (left)
Western blot
results. Shows
positive control
only, an antibody
for the myosin gene
(blue band).
Figure 3 (right)
GFP expression
was nearly identical
in RNAi and
non-RNAi worms,
each with
approximately 3
non-glowing worms
and 8 glowing
worms.
L4 stage CL2070 C.
elegans were
transferred to RNAi
plates and WT plates
Caffeine was
introduced & worms
were soaked for 1 hour
in a 30mM caffeine
solution
After 2 days Worms were washed of caffeine
and returned to original plates
GFP fluorescence was
observed under a dissecting
scope with green light
Movement and speed were
observed and quantitatively
recorded
Worm proteins were
extracted and used for a
Bradford assay,
SDS-PAGE, and a
western immunoblotCaffeine
Table 1 Chi-square analysis for speed phenotype with 1
degree of freedom gave P-value < 0.00001. Value on
left is observed; right is expected. Chi-square = 25.92.