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12/14/16
MOL Lab 6
Introduction
In this lab, we are exploring the effects of RNAi on a specific gene in
the C elegans worm. Our plasmid of interest was the twitchin gene, which
encodes for a protein responsible for regulating muscle contraction and
relaxation in C elegans movement. RNA interference is a technique used to
inhibit gene expression or translation by neutralizing targeted mRNA
molecules. Endogenous double stranded RNA is introduced into the cell and
is cleaved by Dicer into short double-stranded fragments of around 20
nucleotide siRNAs. Each siRNA is unwound into two single-stranded RNAs,
the passenger strand and the guide strand. Once these RNA sequences are
unwound, the guide strand is incorporated into a RISC complex. Finally, this
RISC complex looks for its complementary mRNA sequence to which it
cleaves and degrades. Because the twitchin gene encodes for a protein that
is vital for the movement of the C elegans worm, by knocking down
expression of this protein via RNAi, we expect to see inhibition in movement.
Specifically, we predict a decrease in body bends in response to a touch
stimulus.
Methods
Results
On the first day, we primarily observed young adult worms on both the
control and the RNAi plate and the morphology of both of the worms
appeared to be similar. We conducted 10 trials of touch stimuli and recorded
the number of body bends in response to these stimuli. We defined a body
bend to an inversion of a segment of the C elegans body (i.e. when the S
shaped body turned into a backwards S). For the control C. elegans, we
observed an average of 7.3 body bends per 10 secs, while for the RNAi C
Discussion
Because the twitchin gene is integral to the C elegans movement, we
predicted that the RNAi would interfere with the movement in the worm in
comparison to the control. The results from Table 1 clearly show this
difference as we see that the young adult RNAi C elegans averaged only 3.2
body bends per 10 s after a stimulus in comparison to the young adult
controls 7.3 body bends. This would support the idea that twitchin is
important in the contraction and relaxation in C elegans worms, which led to
the decreased body bends in the RNAi worm. The next day, we observed
mature C elegans and again we see a decrease in body bends for the RNAi
worm in comparison to the control worms: 3.1 body bends to 5.7 body bends
per 10 secs (Table 2). Interestingly, the average body bend rate of the
mature control worms went down in comparison to the control young adult
worms (Graph 1). One hypothesis for this decrease in body bends is that the
worms on the plates were running out of food and were dying, but it is
Trial 1
10
Avg.
Control
11
10
11
7.3
RNAi B Plasmid
3.2
Table 1: Table 1 shows the average body bends/10 secs of both the control
and the RNAi B plasmid worm on Day 1. One can observe a difference in
body bend count in the two constructs (7.3 vs 3.2 body bends/10sec).
Trial 1
10
Avg.
Control
5.7
RNAi B Plasmid
3.1
Table 2: Table 2 shows the average body bends/10 secs of both the control
and the RNAi B plasmid worm on Day 2. One can observe a slightly smaller
difference in body bend count in the two constructs (5.7 vs 3.1 body
bends/10sec).
Graph 1: This graph shows the average body bend movements per 10 sec. of
the control and RNAi construct worms on Days 1 and 2.