Brian Song

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

Initially, we transformed a plasmid containing C. elegans gene into

HT115 bacteria. The (+) plasmid construct contained our plasmid of interest
(plasmid B: twitchin), while the (-) plasmid construct served as a negative
control, as it contained water instead of DNA. Both of these constructs were
transformed into bacteria and this liquid culture of bacteria served as food
for the C. elegans worms. Literature has proven that feeding this dsRNA to
worms produces an effective RNAi effect and can silence gene expression.
After the overnight incubation of the liquid culture, the bacteria were seeded
on NGM plates and then C elegans eggs were subsequently added. Finally,
we observed the effects of the RNAi on the C elegans worms after 2 days of
growth. To observe the effects of the RNAi on the twitchin gene, we
measured the number of body bends per 10 secs in response to a touch
stimuli in both the control and the RNAi knockdown C. elegans worms under
a stereo dissection microscope.

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

elegans, we observed an average of 3.2 (Table 1). We believe this to be a

statistically significant difference in body bends. The next day, we observed
body bends of the two C elegans constructs, and we noticed that there were
mostly mature C elegans and their eggs on the plate. For the control C
elegans, we observed an average of 5.7 body bends per 10 secs, and for the
RNAi construct we only observed an average of 3.1 (Table 2). Again, we
believe this difference to be statistically different.

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

possible that in order to obtain more accurate results, it would be better to

count more than 10 trials per condition as the data set could be skewed by
anomalous movement from a single worm. Additionally, to increase accuracy
in further experiments, I believe that it would be helpful to include a GFP tag
in the plasmid gene so that we can analyze the transformation efficiency into
HT115. Thus, HT115 that fluoresce green indicate that they have
successfully received the transformation. This tag should ensure that only
properly transformed DNA are being introduced to the C elegans worm.

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).

Comparison of Body Bend Averages across 2 Days

8
7
6
5
4
3
2
1
0

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.

I pledge my honor that this work is mine in accordance with University

policies.
/s/ Brian Song