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Quorum Sensing Stephanie Lo, 23 July 07

Quorum Sensing

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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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Page 1: Quorum Sensing

Quorum Sensing

Stephanie Lo, 23 July 07

Page 2: Quorum Sensing

Review

Page 3: Quorum Sensing

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

Page 4: Quorum Sensing

Biobricks

S03623

S03608

I13507

I13507

Senders

Receiver

T9002

Page 5: Quorum Sensing
Page 6: Quorum Sensing

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

Page 7: Quorum Sensing

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)

Page 8: Quorum Sensing

Correlation of RFP to Sender (SI) growth over small OD range

What we expect to see

RFP (RFU)

OD

Page 9: Quorum Sensing

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

Page 10: Quorum Sensing

RFP versus time …

-2000

-1500

-1000

-500

0

500

1000

1500

2000

0 10 20 30 40 50 60

Experiment A

Page 11: Quorum Sensing

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

Page 12: Quorum Sensing

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

Page 13: Quorum Sensing

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

Page 14: Quorum Sensing

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

Page 15: Quorum Sensing

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

Page 16: Quorum Sensing

Detecting a Quorum System Against a Control

GF

P f

luor

esce

nce

per

OD

time

Quorum system

Noninduced (actual)

Noninduced (ideal)

Page 17: Quorum Sensing

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

Page 18: Quorum Sensing

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

Page 19: Quorum Sensing

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

Page 20: Quorum Sensing

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

Page 21: Quorum Sensing

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

Page 22: Quorum Sensing

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

Page 23: Quorum Sensing

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

Page 24: Quorum Sensing

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

Page 25: Quorum Sensing

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

Page 26: Quorum Sensing

Preliminary Experiences with Microscope