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Quorum Sensing. Stephanie Lo, 23 July 07. Review. Some Lessons Learned. Growth of bacteria only minimally affected by plate reader cover; much less drying out Condensation = problem for accuracy? Random wavelengths to measure growth did not work 360/420; 790/850. Biobricks. Senders. - PowerPoint PPT Presentation
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Quorum Sensing
Stephanie Lo, 23 July 07
Review
Some Lessons Learned
• Growth of bacteria only minimally affected by plate reader cover; much less drying out– Condensation = problem for accuracy?
• Random wavelengths to measure growth did not work– 360/420; 790/850
Biobricks
S03623
S03608
I13507
I13507
Senders
Receiver
T9002
Experiment Details
• Grew up liquid cultures of overnight sender (SI) and receiver (T02)
• Dilution with LB to same OD
• Innoculate with different dilutions of sender to receiver
• Manually use plate reader to measure fluorescence (RFP and GFP) and OD
• Shake plate at 37 degrees between reads
Specifics
• Experiment A and B were run manually, C was overnight
• Initial ODs around 0.5 (Experiment A and C) and 0.25 (Experiment B)
Correlation of RFP to Sender (SI) growth over small OD range
What we expect to see
RFP (RFU)
OD
RFP (RFU) versus OD
-1000
-500
0
500
1000
1500
2000
0.475 0.495 0.515 0.535 0.555 0.575 0.595 0.615 0.635 0.655 0.675
OD
RFP
(RFU
)
200 SI
180 SI
160 SI
140 SI
120 SI
100 SI
Experiment A
RFP versus time …
-2000
-1500
-1000
-500
0
500
1000
1500
2000
0 10 20 30 40 50 60
Experiment A
OD versus Time
0.4
0.45
0.5
0.55
0.6
0.65
0.7
0 10 20 30 40 50 60
time (mins)
OD
Experiment A
GFP fluorescence vs time
0
100
200
300
400
500
600
700
800
900
1000
0 20 40 60 80 100 120 140
time (min)
RF
U
10to90
20to80
30to70
40to60
50to50
60to40
70to30
80to20
90to10
(sender to reciever dilutions)
Experiment B
RFP versus time
0
500
1000
1500
2000
2500
3000
3500
0 1 2 3 4 5 6 7 8 9 10
time (hr)
RF
P (
RF
U)
10
20
30
50
60
70
80
Experiment CNote the time lag until substantial RFP
Fluorescence vs. Timeovernight, 7/16
0
500
1000
1500
2000
2500
0 2 4 6 8 10 12 14
time
RF
U (
RF
P)
0
100
200
300
400
500
600
RF
U (
GF
P)
S08-I+T02 RFP
S08-I+T02 GFP
7/16
Initial OD = 0.055
What we’d like to see – Quorum Activity
OHHL concentration /
Sender (SI) amount added /
RFP fluorescence
GF
P f
luor
esce
nce
per
rece
iver
(T
02)
cell
Detecting a Quorum System Against a Control
GF
P f
luor
esce
nce
per
OD
time
Quorum system
Noninduced (actual)
Noninduced (ideal)
GFP Fluorescence vs. Time
0
200
400
600
800
1000
1200
0 10 20 30 40 50 60
time (min)
GF
P (
RF
U)
200 T
20 T
40 T
60 T
80 T
100 T
120 T
140 T
160 T
180 T
Experiment A
GFP versus Sender Added
0
1
2
3
4
5
6
7
20 40 60 80 100 120 140 160 180
SI added
GF
P (
RF
U)
per
T
At start One hour
Experiment A
GFP Fluorescence per Sender Normalized for Number of Receiver Cells
-1
0
1
2
3
4
5
6
7
0 10 20 30 40 50 60 70 80 90
Amount of Sender Added
GF
P (
RF
U)
0 min
60 min
120 min
Experiment B
GFP per Receiver Cell at One Hour
0
2
4
6
8
10
12
14
10 20 30 40 50 60 70 80 90
Sender added
GF
P (
RF
U)
Experiment C
Absolute GFP vs Time
0
500
1000
1500
2000
2500
3000
0 1 2 3 4 5 6
time (hr)
GF
P
Receiver (10)
Receiver (90)
Receiver non-induced
Experiment C
GFP per receiver vs. time
0
5
10
15
20
25
30
35
40
45
0 1 2 3 4 5 6
time (hr)
GF
P p
er
T
10S added
20S added
30S added
50S added
60S added
70S added
80S added
90S added
Experiment C
Interestingly (and in contrast to JT system) …
GFP versus time
0
500
1000
1500
2000
2500
3000
3500
4000
0 2 4 6 8 10 12
time (hr)
GF
P (
RF
U)
90T w ith Sender
90 T w ith 2.5uM OHHL
JT Induced versus Noninduced over time
0
500
1000
1500
2000
2500
3000
3500
0 2 4 6 8 10 12 14
Time (hours)
GF
P (
RF
U)
JT alone
JT w/ 100nM OHHL
JT w/ 10nM OHHL
7/16
In the future: Characterize JT system as well?
• Dilute samples to maintain relatively constant ODs / number of cells
• Determine percent fluorescent through FACS or fluorescence microscope
ODP
erce
ntag
e ce
lls
fluor
esce
nt
Preliminary Experiences with Microscope