"
Team:TU-Delft/test2
From 2012.igem.org
Analysis of Growth retention by addition of
alpha pheromone
Introduction:
Alpha
pheromone is a compound which induces the mating response in S. cerevisae. Promoter Fus1 is induced through a downstream
cascade. Wildtype S. cerevisae
and Δfar1 S. cerevisae are analyzed on growth
rate when alpha pheromone is added.
Method:
Yeasts are
inoculated O/N and harvested in the exponential growth phase. X cells/well are put into a 96 wells plate and a concentration gradient
of alpha pheromone over the wells. A fluorometer
tracks the cell density by measuring the OD600.
Results:
First
background signal is substracted from the OD600
data. The OD600, t=0 gives an indication of
the initial cell density. For the wildtype yeast this
is summarized in table 1. Here can be seen that the difference between the
initial OD and the OD at the end of the measurement of measurement D (9 and 10)
of the wildtype, is relatively low in comparison to
others. This is also seen for measurement F (9 and 10) of the Δfar1
strain.
|
|
|
Wildtype S. cerevisae |
|
|
Δfar1 S. cerevisae |
|
|
|
|
Well |
OD600, t=0 |
OD600, tend=12:00 |
dOD600/dt |
OD600, t=0 |
OD600, tend=12:00 |
dOD600/dt |
|
0 nM |
A9 |
-0,013 |
0,002 |
0,015 |
-0,012 |
0,005 |
0,017 |
|
|
A10 |
-0,011 |
0,006 |
0,017 |
-0,012 |
0,008 |
0,02 |
|
2 nM |
B9 |
-0,01 |
0,008 |
0,018 |
-0,017 |
-0,001 |
0,016 |
|
|
B10 |
-0,009 |
0,012 |
0,021 |
-0,015 |
-0,001 |
0,014 |
|
20 nM |
C9 |
-0,002 |
0,012 |
0,014 |
-0,003 |
0,008 |
0,011 |
|
|
C10 |
0,002 |
0,016 |
0,014 |
-0,005 |
0,01 |
0,015 |
|
200 nM |
D9 |
-0,001 |
0,007 |
0,008 |
-0,008 |
0,004 |
0,012 |
|
|
D10 |
-0,001 |
0,009 |
0,01 |
-0,004 |
0,006 |
0,01 |
|
2 μM |
E9 |
0,005 |
0,017 |
0,012 |
0,002 |
0,016 |
0,014 |
|
|
E10 |
0,003 |
0,02 |
0,017 |
0,001 |
0,016 |
0,015 |
|
20 μM |
F9 |
0,004 |
0,016 |
0,012 |
0,003 |
0,012 |
0,009 |
|
|
F10 |
0,006 |
0,019 |
0,013 |
0,002 |
0,006 |
0,004 |
The data is
averaged (duplo measurement. This give growth curves
according to figure 1 and 2.
In the
figures can be seen that the cells where at the end of their exponential phase,
going into the starving phase (0 nM curve), starting
at ~(t= 0:30:00). Therefore alpha pheromone effect can
only be observed in growth curve in the first 30 minutes, too short to observe
growth retention.
Discussion:
Growth
retention can be observed in wildtype yeast when
alpha pheromone is added. Also correlation in the first 4 hours between the
alpha pheromone concentration and the growth retention can be observed for measurements
B, C, E and F. Measurement D does not follow this correlation but this can be
due too low OD600 values. The Δfar1 strain of S. cerevisae also seems to be affected by addition of alpha
pheromones, but to a lower extent. This can be seen in relatively low
differences of growth
