Igem 2008 Calgary Wetware Full)
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Quorum-coupled Bacteriocin Release: Engineering a Champion
1
sch
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Biostuds
-2 AI
Bad
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Guy #2
☠
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Co
De
De
Cham p
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
ion
De
tec
t
☠
lE3 o C
roy t s e
D
Discharge, Detect, & Destroy
Thane Kubik Kevin McLeod Dave Curran Kingsley Nze
Wetware
Discharge, Detect, & Destroy
d
Ba
1 # y Gu
Cham
pion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Discharge, Detect, & Destroy
rge
cha
Dis
d
Ba
1 # y Gu
Cham
pion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Discharge, Detect, & Destroy
De
tec
t
rge
cha
Dis
d
Ba
1 # y Gu
Cham
pion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Discharge, Detect, & Destroy
De
tec
t
rge
cha
De str oy
Dis
d
Ba
1 # y Gu
Cham
pion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Discharge, Detect, & Destroy
rge
cha
Dis
d
Ba
1 # y Gu
Bad
De
tec
t
#2 Guy
De str oy
Cham
pion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Discharge, Detect, & Destroy
Bad
Di
rge
sch arg e
cha
Dis
d
Ba
1 # y Gu
De
tec
t
#2 Guy
De str oy
Cham
pion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Discharge, Detect, & Destroy
Bad
Di
rge
sch arg e
cha
Dis
d
Ba
1 # y Gu
De
tec
t
#2 Guy
De str oy
Cham
pion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
De
tec
t
Discharge, Detect, & Destroy
Wetware
Discharge, Detect, & Destroy
Bad
Di
rge
sch arg e
cha
Dis
d
Ba
1 # y Gu
De
tec
t
#2 Guy
De str oy
Cham
pion
De
tec
t
oy r t es
D
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Discharge, Detect, & Destroy
-2
sch arg e AI
De
tec
t
#2 Guy
De str oy
Bad
Di
rge
L AH
cha
Dis
d
Ba
1 # y Gu
Cham
pion
De
tec
t
oy r t es
D
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Discharge, Detect, & Destroy
-2
sch arg e AI
De
tec
t
#2 Guy
De str oy
Bad
Di
rge
L AH
cha
Dis
d
Ba
1 # y Gu
Cham
pion
De
tec
t
oy r t es
D
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Discharge, Detect, & Destroy
#2 Guy
-2
sch arg e AI
Bad
Di
rge
L AH
cha
Dis
d
Ba
1 # y Gu
De
tec
t
☠
De Col str E2 oy
Cham
pion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
De
tec
t
Discharge, Detect, & Destroy
lo E3 C oy r t s e D
☠
Wetware
What is quorum sensing? Talk nerd to m y e!
Microbial chatter
k l a t a i r e t c a b w o H r e h t o h c to ea
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Number of cells (log)
Quorum sensing: establishing a consensus
Time
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Number of cells (log)
Quorum sensing: establishing a consensus
Time
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Number of cells (log)
Quorum sensing: establishing a consensus
Time
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Number of cells (log)
Quorum sensing: establishing a consensus
Time
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Number of cells (log)
Quorum sensing: establishing a consensus
Time
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Number of cells (log)
Quorum sensing: establishing a consensus
Time
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Do bacteria have anything interesting to say, Doctor??
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Do bacteria have anything interesting to say, Doctor?? Talk of virulence
•
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Vibrio cholerae
Discharge, Detect, & Destroy
Wetware
Do bacteria have anything interesting to say, Doctor?? Talk of virulence
•
Vibrio cholerae
Decisions of biofilm formation
•
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Pseudomonas aeruginosa
Discharge, Detect, & Destroy
Wetware
Do bacteria have anything interesting to say, Doctor?? Talk of virulence
•
Vibrio cholerae
Decisions of biofilm formation
•
Pseudomonas aeruginosa
Dialogue of genetic competence
•
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Streptococcus pneumoniae
Discharge, Detect, & Destroy
Wetware
Do bacteria have anything interesting to say, Doctor?? Talk of virulence
•
Vibrio cholerae
Decisions of biofilm formation
•
Pseudomonas aeruginosa
Dialogue of genetic competence
•
Streptococcus pneumoniae
Chattering with human host
•
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Enterococcus faecalis
Discharge, Detect, & Destroy
Wetware
V. harveyi - sending mixed messages Two Quorum Sensing systems
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
V. harveyi - sending mixed messages Two Quorum Sensing systems
AHL
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
V. harveyi - sending mixed messages Two Quorum Sensing systems
AHL
Intimate conversations
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
V. harveyi - sending mixed messages Two Quorum Sensing systems
AHL
AI-2
Intimate conversations
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
V. harveyi - sending mixed messages Two Quorum Sensing systems
AHL
AI-2
Intimate conversations
Communal living
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
System #1: Champion vs. Bad Guy#1
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Se nd
er
Autoinducer-1 (AI-1/AHL) Signaling in V. harveyi
Receiv er
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Autoinducer-1 (AI-1/AHL) Signaling in V. harveyi
Se nd
er
LuxR
Receiv er
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxR
Se nd
er
AH L
Autoinducer-1 (AI-1/AHL) Signaling in V. harveyi
Receiv er
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AH L
Autoinducer-1 (AI-1/AHL) Signaling in V. harveyi
Se nd
er
LuxR
Receiv er
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AH L
Se nd
er
Autoinducer-1 (AI-1/AHL) Signaling in V. harveyi
LuxR
Receiv er
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AH L
Se nd
er
Autoinducer-1 (AI-1/AHL) Signaling in V. harveyi
LuxR
Receiv er
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Champion vs. Bad Guy#1
Champ
ion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Champion vs. Bad Guy#1
LuxR
Champ
ion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxR
Ba
d
Gu
y#
1
Champion vs. Bad Guy#1
Champ
ion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxR
Ba
d
Gu
y#
1
AH L
Champion vs. Bad Guy#1
Champ
ion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AH L
LuxR
Ba
d
Gu
y#
1
Champion vs. Bad Guy#1
Champ
ion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AH L
Gu
y#
1
Champion vs. Bad Guy#1
Ba
d
LuxR
Champ
ion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AH L
Gu
y#
1
Champion vs. Bad Guy#1
Ba
d
LuxR
Champ
ion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AH L
Gu
y#
1
Champion vs. Bad Guy#1
Ba
d
LuxR
Champ
C
I
L
ion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AH L
Champion vs. Bad Guy#1
LuxR
Champ
C
I
L
ion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Does ColicinE2 kill?
1
2
3
4
#
Spot
1 ColE2 in BL21 2 ColE2 in BL21 + MytC (2hr) 3 ColE2 in BL21 + MytC (4hr) 4 LB + MytC UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Does ColicinE2 kill?
1
2
1
2
3
4
3
4
#
Spot
#
Spot
1 ColE2 in BL21
1 ColE2 in TOP10
2 ColE2 in BL21 + MytC (2hr) 3 ColE2 in BL21 + MytC (4hr) 4 LB + MytC
2 ColE2 in BL21 + MytC (2hr) 3 ColE2 in BL21 + MytC (4hr) 4 LB + MytC
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Can Bad Guy #1 discharge AHL?
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Can Champion detect?
#
Spot
1
2
1
Bad Guy #1
3
4
2 3 4
AHL AHL Bad Guy #1
Discharge, Detect, & Destroy
Wetware
Can Champion detect and destroy?
•
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Does Bad Guy #1 induce Champion to express GFP and release ColE2?
Discharge, Detect, & Destroy
Wetware
Can Champion detect and destroy?
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
•
Does Bad Guy #1 induce Champion to express GFP and release ColE2?
•
Concentration-dependent response?
Discharge, Detect, & Destroy
Wetware
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Champ
Champ
Champ
Champ
Champ
Champ
Champ
Champ
1
2
3
4
5
6
7
8
Discharge, Detect, & Destroy
Wetware
Number of cells (log) UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Champ
Champ
Champ
Champ
Champ
Champ
Champ
Champ
Time
1
2
3
4
5
6
7
8
Discharge, Detect, & Destroy
Wetware
Number of cells (log)
AHL
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Champ
Champ
Champ
Champ
Champ
Champ
0.75 µM 1.25 µM 2.5 µM
Champ
0.5 µM
Champ
Time
1
2
3
4
5
6
7
8
Discharge, Detect, & Destroy
Wetware
Number of cells (log)
AHL
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Champ Champ
Champ Champ
1/3
Champ Champ
1/6
Champ Champ
1/15
Champ
1/30
Champ
0.75 µM 1.25 µM 2.5 µM
Champ
0.5 µM
Champ
Time
BG#1
1
2
3
4
5
6
7
8
Discharge, Detect, & Destroy
Wetware
AHL
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Champ Champ
Champ Champ
Champ Champ
1/3
Champ Champ
1/6
Champ
1/15
Champ
1/30
Champ
0.75 µM 1.25 µM 2.5 µM
Champ
0.5 µM
BG#1
1
2
3
4
5
6
7
8
Discharge, Detect, & Destroy
Wetware
AHL
Champ Champ
Champ Champ
Champ Champ
1/3
Champ Champ
1/6
Champ
1/15
Champ
1/30
Champ
0.75 µM 1.25 µM 2.5 µM
Champ
0.5 µM
BG#1
1
2
3
4
5
6
7
8
Samples taken: 1 hour post induction 2 hours post induction 4 hours post induction
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AHL
Champ Champ
Champ Champ
Champ Champ
1/3
Champ Champ
1/6
Champ
1/15
Champ
1/30
Champ
0.75 µM 1.25 µM 2.5 µM
Champ
0.5 µM
BG#1
1
2
3
4
5
6
7
8
Samples taken: 1 hour post induction 2 hours post induction 4 hours post induction lawn of BG#1 UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AHL
Champ Champ
Champ Champ
Champ Champ
1/3
Champ Champ
1/6
Champ
1/15
Champ
1/30
Champ
0.75 µM 1.25 µM 2.5 µM
Champ
0.5 µM
BG#1
1
2
3
4
5
6
7
8
Samples taken: 1 hour post induction 2 hours post induction 4 hours post induction
1 hr
2 hr
4 hr
C
lawn of BG#1 UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
... and the Champion prevails
0.5 µM AHL
0.75 µM AHL
1.25 µM AHL
2.5 µM AHL
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
... and the Champion prevails
0.5 µM AHL
0.75 µM AHL
BG#1/CC = 1/30
BG#1/CC = 1/15
1.25 µM AHL
2.5 µM AHL
BG#1/CC = 1/3
BG#1/CC = 1/6
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Our Champion in action
BG#1/CC = 1/30
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
BG#1/CC = 1/30
Discharge, Detect, & Destroy
Wetware
System #2: Champion vs. Bad Guy#2
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
LuxR mRNA LuxO
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
P LuxU
LuxR mRNA LuxO
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
P LuxU
LuxR mRNA LuxO
P LuxO
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
P LuxU
LuxR mRNA
P LuxO LuxO
P LuxO
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
P LuxU
LuxR mRNA
P LuxO 54 LuxO
P LuxO
σ
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
P LuxU
LuxR mRNA LuxO
P LuxO
P LuxO 54
σ
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
P LuxU
LuxR mRNA LuxO
P LuxO
P LuxO 54
sRNA
σ
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
P LuxU
LuxR mRNA LuxO
P LuxO
P LuxO 54
sRNA
σ
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
P LuxU
LuxR mRNA LuxO
P LuxO
P LuxO 54
sRNA
σ
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AI
AI-2
AI-2
-2
AI-2
AI-2 2 AI-
AI-2
AI-2
LuxP LuxQ
AI-2
LuxU
P LuxU
LuxR mRNA
AI-2
LuxO
P LuxO
P LuxO 54
sRNA
σ
AI-2
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AI
AI-2
AI-2
-2
AI-2
2 AI-
AI-2
AI-2
AI-2 LuxP LuxQ
AI-2
LuxU
P LuxU
LuxR mRNA
AI-2
LuxO
P LuxO
P LuxO 54
sRNA
σ
AI-2
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AI
AI-2
AI-2
-2
AI-2
2 AI-
AI-2
AI-2
AI-2 LuxP LuxQ
AI-2
LuxU
P LuxU
P
AI-2
LuxO
LuxR mRNA P LuxO
P LuxO 54
sRNA
σ
AI-2
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AI
AI-2
AI-2
-2
AI-2
2 AI-
AI-2
AI-2
AI-2 LuxP LuxQ
AI-2
LuxU
P LuxU
P
AI-2
LuxO
LuxR mRNA P LuxO
P LuxO 54
sRNA
σ
P
AI-2
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AI
AI-2
AI-2
-2
AI-2
2 AI-
AI-2
AI-2
AI-2 LuxP LuxQ
AI-2
LuxU
P LuxU
P
AI-2
LuxO
LuxR mRNA P LuxO
sRNA
P
AI-2
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AI
AI-2
AI-2
-2
AI-2
2 AI-
AI-2
AI-2
AI-2 LuxP LuxQ
AI-2
LuxU
P LuxU
P
AI-2
LuxO
LuxR mRNA P LuxO
sRNA
P
AI-2
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AI
AI-2
AI-2
-2
AI-2
2 AI-
AI-2
AI-2
AI-2 LuxP LuxQ
AI-2
LuxU
P LuxU
P
AI-2
LuxO
LuxR mRNA P LuxO
P
AI-2
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AI
AI-2
AI-2
-2
AI-2
AI-2
2 AI-
AI-2 AI-2 LuxP LuxQ
AI-2
LuxU
P LuxU
P
AI-2
LuxO
LuxR mRNA P LuxO
LuxR
P
AI-2
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AI
AI-2
AI-2
-2
AI-2
AI-2
2 AI-
AI-2 AI-2 LuxP LuxQ
AI-2
LuxU
P LuxU
P
AI-2
LuxO
LuxR mRNA P LuxO
P LuxR AI-2
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AI
AI-2
AI-2
-2
AI-2
AI-2
2 AI-
AI-2 AI-2 LuxP LuxQ
AI-2
LuxU
P LuxU
P
AI-2
LuxO
LuxR mRNA P LuxO
P LuxR AI-2
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Why is AI-2 signaling worth all of the blood, sweat and tears??
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Why is AI-2 signaling worth all of the blood, sweat and tears?? Over 50% bacteria (i.e. >300 species) whose genome has been sequenced encode LuxS (required for AI-2 production)
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Why is AI-2 signaling worth all of the blood, sweat and tears?? Over 50% bacteria (i.e. >300 species) whose genome has been sequenced encode LuxS (required for AI-2 production) Genes controlled by AI-2 signaling: •E. coli OI57:H7: Virulence, motility, secretion •Streptococcus pneumoniae: Competence,Virulence, Persistence •Vibrio cholera: Virulence, Biofilms, Toxins
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Why is AI-2 signaling worth all of the blood, sweat and tears?? Over 50% bacteria (i.e. >300 species) whose genome has been sequenced encode LuxS (required for AI-2 production) Genes controlled by AI-2 signaling: •E. coli OI57:H7: Virulence, motility, secretion •Streptococcus pneumoniae: Competence,Virulence, Persistence •Vibrio cholera: Virulence, Biofilms, Toxins Addition of a second signaling system to the Registry
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Why is AI-2 signaling worth all of the blood, sweat and tears?? Over 50% bacteria (i.e. >300 species) whose genome has been sequenced encode LuxS (required for AI-2 production) Genes controlled by AI-2 signaling: •E. coli OI57:H7: Virulence, motility, secretion •Streptococcus pneumoniae: Competence,Virulence, Persistence •Vibrio cholera: Virulence, Biofilms, Toxins Addition of a second signaling system to the Registry “If you can’t build it, you can’t fully understand it!!” (Further Insight into AI-2 signaling)
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
LuxO
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
P LuxU
LuxO
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
LuxO
P LuxU
P LuxO
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
P LuxU
P LuxO LuxO
P LuxO
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
P LuxU
P LuxO 54
σ
LuxO
P LuxO
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
LuxO
P LuxU
P LuxO
P LuxOσ54
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
LuxO
P LuxU
P LuxO
P LuxOσ54
R
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
LuxO
P LuxU
P LuxO
P LuxOσ54
R
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
LuxO
P LuxU
P LuxO
P LuxOσ54
R
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Bad Guy #2
LuxP LuxQ LuxU
LuxO
P LuxU
P LuxO
P LuxOσ54
R
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Bad Guy #2
AI
-2
AI-2 AI-2
AI-2
2
AILuxP LuxQ
AI-2
LuxU
AI-2
P LuxU AI-2
LuxO
P LuxO
P LuxOσ54
R
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Bad Guy #2
AI
-2
AI-2
AI-2
2
AILuxP LuxQ
AI-2 AI-2
LuxU
AI-2
P LuxU AI-2
LuxO
P LuxO
P LuxOσ54
R
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Bad Guy #2
AI
-2
AI-2
AI-2
2
AILuxP LuxQ
AI-2 AI-2
LuxU
P LuxU AI-2
P LuxO
AI-2
P LuxO
P LuxOσ54
R
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Bad Guy #2
AI
-2
AI-2
AI-2
2
AILuxP LuxQ
AI-2 AI-2
LuxU
P LuxU AI-2
P LuxO
AI-2
P LuxO
P
P LuxOσ54
R
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Bad Guy #2
AI
-2
AI-2
AI-2
2
AILuxP LuxQ
AI-2 AI-2
LuxU
P LuxU AI-2
P LuxO
AI-2
P LuxO
P
R
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Bad Guy #2
AI
-2
AI-2
AI-2
2
AILuxP LuxQ
AI-2 AI-2
LuxU
P LuxU AI-2
P LuxO
AI-2
P LuxO
P
R
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Bad Guy #2
AI
-2
AI-2
AI-2
2
AILuxP LuxQ
AI-2 AI-2
LuxU
P LuxU AI-2
P LuxO
AI-2
P LuxO
P
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Bad Guy #2
AI
-2
AI-2
AI-2
2
AILuxP LuxQ
AI-2 AI-2
LuxU
P LuxU AI-2
P LuxO
AI-2
P LuxO
P
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Bad Guy #2
AI
-2
AI-2
AI-2
2
AILuxP LuxQ
AI-2 AI-2
LuxU
AI-2
P LuxU AI-2
P LuxO
P LuxO
P
C
I
L
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Bad Guy #2
AI
-2
AI-2
AI-2
2
AILuxP LuxQ
C
AI-2 AI-2
LuxU
AI-2
P LuxU AI-2
P LuxO
P LuxO
P
C
I
L
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AI
-2
AI-2
AI-2
2
AILuxP LuxQ
AI-2 AI-2
LuxU
AI-2
P LuxU AI-2
P LuxO
P LuxO
P
C
I
L
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Engineering the Champion
LuxP
AI-2
LuxQ
P
LuxU
Signaling
LuxU
P LuxO
P
P
LuxO
Response
Champion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Signaling circuit
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Signaling circuit
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Signaling circuit
PCR amplification
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Signaling circuit
PCR amplification
TOPO Cloning
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Signaling circuit
PCR amplification
TOPO Cloning
Verification by Colony PCR/ Restriction Digest/Sequencing
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Signaling circuit
PCR amplification
TOPO Cloning
Verification by Colony PCR/ Restriction Digest/Sequencing
PCR amplification with BioBrick primers
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Signaling circuit
PCR amplification
TOPO Cloning
Verification by Colony PCR/ Restriction Digest/Sequencing
PCR amplification with BioBrick primers
BioBrick Cloning and Circuit(s) assembly
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Signaling circuit
PCR amplification
TOPO Cloning
Verification by Colony PCR/ Restriction Digest/Sequencing Mutagenesis PCR amplification with BioBrick primers
BioBrick Cloning and Circuit(s) assembly
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Response circuit
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Testing the Response circuit LuxO D47E
LuxO D47A
mimics LuxO-℗ = active
inactive
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Testing the Response circuit LuxO D47E
LuxO D47A
mimics LuxO-℗ = active
inactive
LuxO
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
P
Discharge, Detect, & Destroy
Wetware
Testing the Response circuit LuxO D47E
LuxO D47A
mimics LuxO-℗ = active
inactive
LuxO
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
P
σ54
Discharge, Detect, & Destroy
Wetware
Testing the Response circuit LuxO D47E
LuxO D47A
mimics LuxO-℗ = active
inactive
P
LuxO 54 σ
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Testing the Response circuit LuxO D47E
LuxO D47A
mimics LuxO-℗ = active
inactive
P
LuxO 54 σ
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
R
Discharge, Detect, & Destroy
Wetware
Testing the Response circuit LuxO D47E
LuxO D47A
mimics LuxO-℗ = active
inactive
P
LuxO 54 σ
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
R
R
Discharge, Detect, & Destroy
Wetware
Testing the Response circuit LuxO D47E
LuxO D47A
mimics LuxO-℗ = active
inactive
P
LuxO 54 σ
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
R
R
Discharge, Detect, & Destroy
Wetware
Testing the Response circuit LuxO D47E
LuxO D47A
mimics LuxO-℗ = active
inactive
P
LuxO 54 σ
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
R
R
Discharge, Detect, & Destroy
Wetware
Testing the Response circuit LuxO D47E
LuxO D47A
mimics LuxO-℗ = active
inactive
P
LuxO 54 σ
R
R
NO RFP, NO ColE3 UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Testing the Response circuit LuxO D47E
LuxO D47A
mimics LuxO-℗ = active
inactive
LuxO
P
LuxO 54 σ
R
R
NO RFP, NO ColE3 UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Testing the Response circuit LuxO D47E
LuxO D47A
mimics LuxO-℗ = active
inactive
LuxO
P
LuxO 54 σ
R
σ54
R
NO RFP, NO ColE3 UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Testing the Response circuit LuxO D47E
LuxO D47A
mimics LuxO-℗ = active
inactive
LuxO
P
LuxO 54 σ
R
R
NO RFP, NO ColE3 UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Testing the Response circuit LuxO D47E
LuxO D47A
mimics LuxO-℗ = active
inactive
LuxO
P
LuxO 54 σ
R
R
NO RFP, NO ColE3 UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Testing the Response circuit LuxO D47E
LuxO D47A
mimics LuxO-℗ = active
inactive
LuxO
P
LuxO 54 σ
R
R
NO RFP, NO ColE3 UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
RFP & ColE3
Discharge, Detect, & Destroy
Wetware
Summary Part
System #1
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
BBk
Sequenced
ColE2 operon
BBk_131000
Yes
ColE2 w/o pSOS
BBk_131009
Yes
AHL inducible ColE2 with GFP
BBk_131010
Functional test
AHL inducible GFP w/o term. (intermediate)
BBk_131026
Functional test
Discharge, Detect, & Destroy
Wetware
Summary Part
System #1
BBk
ColE2 operon
BBk_131000
Yes
ColE2 w/o pSOS
BBk_131009
Yes
AHL inducible ColE2 with GFP
BBk_131010
Functional test
AHL inducible GFP w/o term. (intermediate)
BBk_131026
Functional test
Part
System #2
Sequenced
BBk
Sequenced
LuxPQ, V. harveyi
BBk_131015
Yes*
LuxOU, V. harveyi
BBk_131016
Yes
qrr4 (quorum-reg. RNA-4) promoter, V. harveyi
BBk_131017
Yes
LuxO inducible RFP
BBk_131019
Functional test
LuxO D47E, V. harveyi
BBk_131022
Yes
LuxO D47A, V. harveyi
BBk_131023
Yes
*Silent mutations to remove EcoRI site in LuxP, EcoRI site in LuxQ, XbaI site in LuxQ
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Conclusions and Summary
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Conclusions and Summary
•
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Establishment of Quorum-coupled Bacteriocin Release Proof-of-Principle: System #1 (AHL-induced ColE2 release)
Discharge, Detect, & Destroy
Wetware
Conclusions and Summary
•
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Establishment of Quorum-coupled Bacteriocin Release Proof-of-Principle: System #1 (AHL-induced ColE2 release)
Discharge, Detect, & Destroy
Wetware
Conclusions and Summary
•
Establishment of Quorum-coupled Bacteriocin Release Proof-of-Principle: System #1 (AHL-induced ColE2 release)
•
Elements required for construction of V. harveyi AI-2 signaling within E. coli deposited in Registry
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Significance
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Significance
•
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Quorum-coupled Bacteriocin Release allows for detection and annihilation of specific Bad Guys based on pheromonal identity
Discharge, Detect, & Destroy
Wetware
Significance
•
Quorum-coupled Bacteriocin Release allows for detection and annihilation of specific Bad Guys based on pheromonal identity
•
Incorporation of Reporter activity allows user to rapidly identify Bad Guy present
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Significance
•
Quorum-coupled Bacteriocin Release allows for detection and annihilation of specific Bad Guys based on pheromonal identity
•
Incorporation of Reporter activity allows user to rapidly identify Bad Guy present
•
AI-2 signaling used by hundreds of bacterial species to coordinate activity within and outside species
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Significance
•
Quorum-coupled Bacteriocin Release allows for detection and annihilation of specific Bad Guys based on pheromonal identity
•
Incorporation of Reporter activity allows user to rapidly identify Bad Guy present
•
AI-2 signaling used by hundreds of bacterial species to coordinate activity within and outside species
•
Quorum Signaling: A new target for antibacterial therapeutics
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Future Directions/Continuing Projects
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Future Directions/Continuing Projects
•
Completion of V. harveyi AI-2 Signaling circuit within E. coli
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Future Directions/Continuing Projects
• •
Completion of V. harveyi AI-2 Signaling circuit within E. coli Selection of species-specific pheromones of pathogenic bacteria (e.g. Cholera Autoinducer-1, CAI-1)
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Future Directions/Continuing Projects
• •
Completion of V. harveyi AI-2 Signaling circuit within E. coli
•
Use of additional bacteriocins (e.g. vibriocins and pyocins)
Selection of species-specific pheromones of pathogenic bacteria (e.g. Cholera Autoinducer-1, CAI-1)
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Acknowledgments
Sonja Georgijevic
Christian Jacob
Bonnie Bassler
Michael Surette
University of Calgary
University of Calgary
Princeton University
University of Calgary
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Questions
d
Ba
1 # y u G
d Ba y#
Gu 2
Cham p
ion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
1
sch
arg e
Biostuds
-2 AI
Bad
Di
L
e arg
ch Dis AH
d Ba
y# u G
Guy #2
☠
lE2
tec
str oy
t
Co
De
De
Cham p
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
ion
De
tec
t
☠
lE3 o C
roy t s e
D
Discharge, Detect, & Destroy
Thane Kubik Kevin McLeod Dave Curran Kingsley Nze
Wetware
Discharge, Detect, & Destroy
d
Ba
1 # y Gu
Cham
pion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Discharge, Detect, & Destroy
rge
cha
Dis
d
Ba
1 # y Gu
Cham
pion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Discharge, Detect, & Destroy
De
tec
t
rge
cha
Dis
d
Ba
1 # y Gu
Cham
pion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Discharge, Detect, & Destroy
De
tec
t
rge
cha
De str oy
Dis
d
Ba
1 # y Gu
Cham
pion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Discharge, Detect, & Destroy
rge
cha
Dis
d
Ba
1 # y Gu
Bad
De
tec
t
#2 Guy
De str oy
Cham
pion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Discharge, Detect, & Destroy
Bad
Di
rge
sch arg e
cha
Dis
d
Ba
1 # y Gu
De
tec
t
#2 Guy
De str oy
Cham
pion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Discharge, Detect, & Destroy
Bad
Di
rge
sch arg e
cha
Dis
d
Ba
1 # y Gu
De
tec
t
#2 Guy
De str oy
Cham
pion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
De
tec
t
Discharge, Detect, & Destroy
Wetware
Discharge, Detect, & Destroy
Bad
Di
rge
sch arg e
cha
Dis
d
Ba
1 # y Gu
De
tec
t
#2 Guy
De str oy
Cham
pion
De
tec
t
oy r t es
D
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Discharge, Detect, & Destroy
-2
sch arg e AI
De
tec
t
#2 Guy
De str oy
Bad
Di
rge
L AH
cha
Dis
d
Ba
1 # y Gu
Cham
pion
De
tec
t
oy r t es
D
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Discharge, Detect, & Destroy
-2
sch arg e AI
De
tec
t
#2 Guy
De str oy
Bad
Di
rge
L AH
cha
Dis
d
Ba
1 # y Gu
Cham
pion
De
tec
t
oy r t es
D
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Discharge, Detect, & Destroy
#2 Guy
-2
sch arg e AI
Bad
Di
rge
L AH
cha
Dis
d
Ba
1 # y Gu
De
tec
t
☠
De Col str E2 oy
Cham
pion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
De
tec
t
Discharge, Detect, & Destroy
lo E3 C oy r t s e D
☠
Wetware
What is quorum sensing? Talk nerd to m y e!
Microbial chatter
k l a t a i r e t c a b w o H r e h t o h c to ea
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Number of cells (log)
Quorum sensing: establishing a consensus
Time
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Number of cells (log)
Quorum sensing: establishing a consensus
Time
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Number of cells (log)
Quorum sensing: establishing a consensus
Time
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Number of cells (log)
Quorum sensing: establishing a consensus
Time
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Number of cells (log)
Quorum sensing: establishing a consensus
Time
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Number of cells (log)
Quorum sensing: establishing a consensus
Time
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Do bacteria have anything interesting to say, Doctor??
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Do bacteria have anything interesting to say, Doctor?? Talk of virulence
•
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Vibrio cholerae
Discharge, Detect, & Destroy
Wetware
Do bacteria have anything interesting to say, Doctor?? Talk of virulence
•
Vibrio cholerae
Decisions of biofilm formation
•
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Pseudomonas aeruginosa
Discharge, Detect, & Destroy
Wetware
Do bacteria have anything interesting to say, Doctor?? Talk of virulence
•
Vibrio cholerae
Decisions of biofilm formation
•
Pseudomonas aeruginosa
Dialogue of genetic competence
•
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Streptococcus pneumoniae
Discharge, Detect, & Destroy
Wetware
Do bacteria have anything interesting to say, Doctor?? Talk of virulence
•
Vibrio cholerae
Decisions of biofilm formation
•
Pseudomonas aeruginosa
Dialogue of genetic competence
•
Streptococcus pneumoniae
Chattering with human host
•
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Enterococcus faecalis
Discharge, Detect, & Destroy
Wetware
V. harveyi - sending mixed messages Two Quorum Sensing systems
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
V. harveyi - sending mixed messages Two Quorum Sensing systems
AHL
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
V. harveyi - sending mixed messages Two Quorum Sensing systems
AHL
Intimate conversations
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
V. harveyi - sending mixed messages Two Quorum Sensing systems
AHL
AI-2
Intimate conversations
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
V. harveyi - sending mixed messages Two Quorum Sensing systems
AHL
AI-2
Intimate conversations
Communal living
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
System #1: Champion vs. Bad Guy#1
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Se nd
er
Autoinducer-1 (AI-1/AHL) Signaling in V. harveyi
Receiv er
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Autoinducer-1 (AI-1/AHL) Signaling in V. harveyi
Se nd
er
LuxR
Receiv er
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxR
Se nd
er
AH L
Autoinducer-1 (AI-1/AHL) Signaling in V. harveyi
Receiv er
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AH L
Autoinducer-1 (AI-1/AHL) Signaling in V. harveyi
Se nd
er
LuxR
Receiv er
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AH L
Se nd
er
Autoinducer-1 (AI-1/AHL) Signaling in V. harveyi
LuxR
Receiv er
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AH L
Se nd
er
Autoinducer-1 (AI-1/AHL) Signaling in V. harveyi
LuxR
Receiv er
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Champion vs. Bad Guy#1
Champ
ion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Champion vs. Bad Guy#1
LuxR
Champ
ion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxR
Ba
d
Gu
y#
1
Champion vs. Bad Guy#1
Champ
ion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxR
Ba
d
Gu
y#
1
AH L
Champion vs. Bad Guy#1
Champ
ion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AH L
LuxR
Ba
d
Gu
y#
1
Champion vs. Bad Guy#1
Champ
ion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AH L
Gu
y#
1
Champion vs. Bad Guy#1
Ba
d
LuxR
Champ
ion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AH L
Gu
y#
1
Champion vs. Bad Guy#1
Ba
d
LuxR
Champ
ion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AH L
Gu
y#
1
Champion vs. Bad Guy#1
Ba
d
LuxR
Champ
C
I
L
ion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AH L
Champion vs. Bad Guy#1
LuxR
Champ
C
I
L
ion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Does ColicinE2 kill?
1
2
3
4
#
Spot
1 ColE2 in BL21 2 ColE2 in BL21 + MytC (2hr) 3 ColE2 in BL21 + MytC (4hr) 4 LB + MytC UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Does ColicinE2 kill?
1
2
1
2
3
4
3
4
#
Spot
#
Spot
1 ColE2 in BL21
1 ColE2 in TOP10
2 ColE2 in BL21 + MytC (2hr) 3 ColE2 in BL21 + MytC (4hr) 4 LB + MytC
2 ColE2 in BL21 + MytC (2hr) 3 ColE2 in BL21 + MytC (4hr) 4 LB + MytC
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Can Bad Guy #1 discharge AHL?
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Can Champion detect?
#
Spot
1
2
1
Bad Guy #1
3
4
2 3 4
AHL AHL Bad Guy #1
Discharge, Detect, & Destroy
Wetware
Can Champion detect and destroy?
•
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Does Bad Guy #1 induce Champion to express GFP and release ColE2?
Discharge, Detect, & Destroy
Wetware
Can Champion detect and destroy?
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
•
Does Bad Guy #1 induce Champion to express GFP and release ColE2?
•
Concentration-dependent response?
Discharge, Detect, & Destroy
Wetware
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Champ
Champ
Champ
Champ
Champ
Champ
Champ
Champ
1
2
3
4
5
6
7
8
Discharge, Detect, & Destroy
Wetware
Number of cells (log) UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Champ
Champ
Champ
Champ
Champ
Champ
Champ
Champ
Time
1
2
3
4
5
6
7
8
Discharge, Detect, & Destroy
Wetware
Number of cells (log)
AHL
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Champ
Champ
Champ
Champ
Champ
Champ
0.75 µM 1.25 µM 2.5 µM
Champ
0.5 µM
Champ
Time
1
2
3
4
5
6
7
8
Discharge, Detect, & Destroy
Wetware
Number of cells (log)
AHL
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Champ Champ
Champ Champ
1/3
Champ Champ
1/6
Champ Champ
1/15
Champ
1/30
Champ
0.75 µM 1.25 µM 2.5 µM
Champ
0.5 µM
Champ
Time
BG#1
1
2
3
4
5
6
7
8
Discharge, Detect, & Destroy
Wetware
AHL
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Champ Champ
Champ Champ
Champ Champ
1/3
Champ Champ
1/6
Champ
1/15
Champ
1/30
Champ
0.75 µM 1.25 µM 2.5 µM
Champ
0.5 µM
BG#1
1
2
3
4
5
6
7
8
Discharge, Detect, & Destroy
Wetware
AHL
Champ Champ
Champ Champ
Champ Champ
1/3
Champ Champ
1/6
Champ
1/15
Champ
1/30
Champ
0.75 µM 1.25 µM 2.5 µM
Champ
0.5 µM
BG#1
1
2
3
4
5
6
7
8
Samples taken: 1 hour post induction 2 hours post induction 4 hours post induction
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AHL
Champ Champ
Champ Champ
Champ Champ
1/3
Champ Champ
1/6
Champ
1/15
Champ
1/30
Champ
0.75 µM 1.25 µM 2.5 µM
Champ
0.5 µM
BG#1
1
2
3
4
5
6
7
8
Samples taken: 1 hour post induction 2 hours post induction 4 hours post induction lawn of BG#1 UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AHL
Champ Champ
Champ Champ
Champ Champ
1/3
Champ Champ
1/6
Champ
1/15
Champ
1/30
Champ
0.75 µM 1.25 µM 2.5 µM
Champ
0.5 µM
BG#1
1
2
3
4
5
6
7
8
Samples taken: 1 hour post induction 2 hours post induction 4 hours post induction
1 hr
2 hr
4 hr
C
lawn of BG#1 UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
... and the Champion prevails
0.5 µM AHL
0.75 µM AHL
1.25 µM AHL
2.5 µM AHL
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
... and the Champion prevails
0.5 µM AHL
0.75 µM AHL
BG#1/CC = 1/30
BG#1/CC = 1/15
1.25 µM AHL
2.5 µM AHL
BG#1/CC = 1/3
BG#1/CC = 1/6
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Our Champion in action
BG#1/CC = 1/30
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
BG#1/CC = 1/30
Discharge, Detect, & Destroy
Wetware
System #2: Champion vs. Bad Guy#2
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
LuxR mRNA LuxO
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
P LuxU
LuxR mRNA LuxO
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
P LuxU
LuxR mRNA LuxO
P LuxO
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
P LuxU
LuxR mRNA
P LuxO LuxO
P LuxO
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
P LuxU
LuxR mRNA
P LuxO 54 LuxO
P LuxO
σ
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
P LuxU
LuxR mRNA LuxO
P LuxO
P LuxO 54
σ
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
P LuxU
LuxR mRNA LuxO
P LuxO
P LuxO 54
sRNA
σ
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
P LuxU
LuxR mRNA LuxO
P LuxO
P LuxO 54
sRNA
σ
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
P LuxU
LuxR mRNA LuxO
P LuxO
P LuxO 54
sRNA
σ
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AI
AI-2
AI-2
-2
AI-2
AI-2 2 AI-
AI-2
AI-2
LuxP LuxQ
AI-2
LuxU
P LuxU
LuxR mRNA
AI-2
LuxO
P LuxO
P LuxO 54
sRNA
σ
AI-2
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AI
AI-2
AI-2
-2
AI-2
2 AI-
AI-2
AI-2
AI-2 LuxP LuxQ
AI-2
LuxU
P LuxU
LuxR mRNA
AI-2
LuxO
P LuxO
P LuxO 54
sRNA
σ
AI-2
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AI
AI-2
AI-2
-2
AI-2
2 AI-
AI-2
AI-2
AI-2 LuxP LuxQ
AI-2
LuxU
P LuxU
P
AI-2
LuxO
LuxR mRNA P LuxO
P LuxO 54
sRNA
σ
AI-2
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AI
AI-2
AI-2
-2
AI-2
2 AI-
AI-2
AI-2
AI-2 LuxP LuxQ
AI-2
LuxU
P LuxU
P
AI-2
LuxO
LuxR mRNA P LuxO
P LuxO 54
sRNA
σ
P
AI-2
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AI
AI-2
AI-2
-2
AI-2
2 AI-
AI-2
AI-2
AI-2 LuxP LuxQ
AI-2
LuxU
P LuxU
P
AI-2
LuxO
LuxR mRNA P LuxO
sRNA
P
AI-2
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AI
AI-2
AI-2
-2
AI-2
2 AI-
AI-2
AI-2
AI-2 LuxP LuxQ
AI-2
LuxU
P LuxU
P
AI-2
LuxO
LuxR mRNA P LuxO
sRNA
P
AI-2
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AI
AI-2
AI-2
-2
AI-2
2 AI-
AI-2
AI-2
AI-2 LuxP LuxQ
AI-2
LuxU
P LuxU
P
AI-2
LuxO
LuxR mRNA P LuxO
P
AI-2
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AI
AI-2
AI-2
-2
AI-2
AI-2
2 AI-
AI-2 AI-2 LuxP LuxQ
AI-2
LuxU
P LuxU
P
AI-2
LuxO
LuxR mRNA P LuxO
LuxR
P
AI-2
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AI
AI-2
AI-2
-2
AI-2
AI-2
2 AI-
AI-2 AI-2 LuxP LuxQ
AI-2
LuxU
P LuxU
P
AI-2
LuxO
LuxR mRNA P LuxO
P LuxR AI-2
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AI
AI-2
AI-2
-2
AI-2
AI-2
2 AI-
AI-2 AI-2 LuxP LuxQ
AI-2
LuxU
P LuxU
P
AI-2
LuxO
LuxR mRNA P LuxO
P LuxR AI-2
Receiver UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Why is AI-2 signaling worth all of the blood, sweat and tears??
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Why is AI-2 signaling worth all of the blood, sweat and tears?? Over 50% bacteria (i.e. >300 species) whose genome has been sequenced encode LuxS (required for AI-2 production)
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Why is AI-2 signaling worth all of the blood, sweat and tears?? Over 50% bacteria (i.e. >300 species) whose genome has been sequenced encode LuxS (required for AI-2 production) Genes controlled by AI-2 signaling: •E. coli OI57:H7: Virulence, motility, secretion •Streptococcus pneumoniae: Competence,Virulence, Persistence •Vibrio cholera: Virulence, Biofilms, Toxins
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Why is AI-2 signaling worth all of the blood, sweat and tears?? Over 50% bacteria (i.e. >300 species) whose genome has been sequenced encode LuxS (required for AI-2 production) Genes controlled by AI-2 signaling: •E. coli OI57:H7: Virulence, motility, secretion •Streptococcus pneumoniae: Competence,Virulence, Persistence •Vibrio cholera: Virulence, Biofilms, Toxins Addition of a second signaling system to the Registry
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Why is AI-2 signaling worth all of the blood, sweat and tears?? Over 50% bacteria (i.e. >300 species) whose genome has been sequenced encode LuxS (required for AI-2 production) Genes controlled by AI-2 signaling: •E. coli OI57:H7: Virulence, motility, secretion •Streptococcus pneumoniae: Competence,Virulence, Persistence •Vibrio cholera: Virulence, Biofilms, Toxins Addition of a second signaling system to the Registry “If you can’t build it, you can’t fully understand it!!” (Further Insight into AI-2 signaling)
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
LuxO
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
P LuxU
LuxO
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
LuxO
P LuxU
P LuxO
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
P LuxU
P LuxO LuxO
P LuxO
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
P LuxU
P LuxO 54
σ
LuxO
P LuxO
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
LuxO
P LuxU
P LuxO
P LuxOσ54
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
LuxO
P LuxU
P LuxO
P LuxOσ54
R
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
LuxO
P LuxU
P LuxO
P LuxOσ54
R
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
LuxP LuxQ LuxU
LuxO
P LuxU
P LuxO
P LuxOσ54
R
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Bad Guy #2
LuxP LuxQ LuxU
LuxO
P LuxU
P LuxO
P LuxOσ54
R
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Bad Guy #2
AI
-2
AI-2 AI-2
AI-2
2
AILuxP LuxQ
AI-2
LuxU
AI-2
P LuxU AI-2
LuxO
P LuxO
P LuxOσ54
R
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Bad Guy #2
AI
-2
AI-2
AI-2
2
AILuxP LuxQ
AI-2 AI-2
LuxU
AI-2
P LuxU AI-2
LuxO
P LuxO
P LuxOσ54
R
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Bad Guy #2
AI
-2
AI-2
AI-2
2
AILuxP LuxQ
AI-2 AI-2
LuxU
P LuxU AI-2
P LuxO
AI-2
P LuxO
P LuxOσ54
R
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Bad Guy #2
AI
-2
AI-2
AI-2
2
AILuxP LuxQ
AI-2 AI-2
LuxU
P LuxU AI-2
P LuxO
AI-2
P LuxO
P
P LuxOσ54
R
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Bad Guy #2
AI
-2
AI-2
AI-2
2
AILuxP LuxQ
AI-2 AI-2
LuxU
P LuxU AI-2
P LuxO
AI-2
P LuxO
P
R
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Bad Guy #2
AI
-2
AI-2
AI-2
2
AILuxP LuxQ
AI-2 AI-2
LuxU
P LuxU AI-2
P LuxO
AI-2
P LuxO
P
R
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Bad Guy #2
AI
-2
AI-2
AI-2
2
AILuxP LuxQ
AI-2 AI-2
LuxU
P LuxU AI-2
P LuxO
AI-2
P LuxO
P
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Bad Guy #2
AI
-2
AI-2
AI-2
2
AILuxP LuxQ
AI-2 AI-2
LuxU
P LuxU AI-2
P LuxO
AI-2
P LuxO
P
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Bad Guy #2
AI
-2
AI-2
AI-2
2
AILuxP LuxQ
AI-2 AI-2
LuxU
AI-2
P LuxU AI-2
P LuxO
P LuxO
P
C
I
L
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Bad Guy #2
AI
-2
AI-2
AI-2
2
AILuxP LuxQ
C
AI-2 AI-2
LuxU
AI-2
P LuxU AI-2
P LuxO
P LuxO
P
C
I
L
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
AI
-2
AI-2
AI-2
2
AILuxP LuxQ
AI-2 AI-2
LuxU
AI-2
P LuxU AI-2
P LuxO
P LuxO
P
C
I
L
Champion UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Engineering the Champion
LuxP
AI-2
LuxQ
P
LuxU
Signaling
LuxU
P LuxO
P
P
LuxO
Response
Champion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Signaling circuit
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Signaling circuit
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Signaling circuit
PCR amplification
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Signaling circuit
PCR amplification
TOPO Cloning
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Signaling circuit
PCR amplification
TOPO Cloning
Verification by Colony PCR/ Restriction Digest/Sequencing
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Signaling circuit
PCR amplification
TOPO Cloning
Verification by Colony PCR/ Restriction Digest/Sequencing
PCR amplification with BioBrick primers
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Signaling circuit
PCR amplification
TOPO Cloning
Verification by Colony PCR/ Restriction Digest/Sequencing
PCR amplification with BioBrick primers
BioBrick Cloning and Circuit(s) assembly
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Signaling circuit
PCR amplification
TOPO Cloning
Verification by Colony PCR/ Restriction Digest/Sequencing Mutagenesis PCR amplification with BioBrick primers
BioBrick Cloning and Circuit(s) assembly
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Response circuit
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Testing the Response circuit LuxO D47E
LuxO D47A
mimics LuxO-℗ = active
inactive
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Testing the Response circuit LuxO D47E
LuxO D47A
mimics LuxO-℗ = active
inactive
LuxO
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
P
Discharge, Detect, & Destroy
Wetware
Testing the Response circuit LuxO D47E
LuxO D47A
mimics LuxO-℗ = active
inactive
LuxO
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
P
σ54
Discharge, Detect, & Destroy
Wetware
Testing the Response circuit LuxO D47E
LuxO D47A
mimics LuxO-℗ = active
inactive
P
LuxO 54 σ
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Testing the Response circuit LuxO D47E
LuxO D47A
mimics LuxO-℗ = active
inactive
P
LuxO 54 σ
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
R
Discharge, Detect, & Destroy
Wetware
Testing the Response circuit LuxO D47E
LuxO D47A
mimics LuxO-℗ = active
inactive
P
LuxO 54 σ
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
R
R
Discharge, Detect, & Destroy
Wetware
Testing the Response circuit LuxO D47E
LuxO D47A
mimics LuxO-℗ = active
inactive
P
LuxO 54 σ
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
R
R
Discharge, Detect, & Destroy
Wetware
Testing the Response circuit LuxO D47E
LuxO D47A
mimics LuxO-℗ = active
inactive
P
LuxO 54 σ
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
R
R
Discharge, Detect, & Destroy
Wetware
Testing the Response circuit LuxO D47E
LuxO D47A
mimics LuxO-℗ = active
inactive
P
LuxO 54 σ
R
R
NO RFP, NO ColE3 UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Testing the Response circuit LuxO D47E
LuxO D47A
mimics LuxO-℗ = active
inactive
LuxO
P
LuxO 54 σ
R
R
NO RFP, NO ColE3 UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Testing the Response circuit LuxO D47E
LuxO D47A
mimics LuxO-℗ = active
inactive
LuxO
P
LuxO 54 σ
R
σ54
R
NO RFP, NO ColE3 UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Testing the Response circuit LuxO D47E
LuxO D47A
mimics LuxO-℗ = active
inactive
LuxO
P
LuxO 54 σ
R
R
NO RFP, NO ColE3 UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Testing the Response circuit LuxO D47E
LuxO D47A
mimics LuxO-℗ = active
inactive
LuxO
P
LuxO 54 σ
R
R
NO RFP, NO ColE3 UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Testing the Response circuit LuxO D47E
LuxO D47A
mimics LuxO-℗ = active
inactive
LuxO
P
LuxO 54 σ
R
R
NO RFP, NO ColE3 UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
RFP & ColE3
Discharge, Detect, & Destroy
Wetware
Summary Part
System #1
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
BBk
Sequenced
ColE2 operon
BBk_131000
Yes
ColE2 w/o pSOS
BBk_131009
Yes
AHL inducible ColE2 with GFP
BBk_131010
Functional test
AHL inducible GFP w/o term. (intermediate)
BBk_131026
Functional test
Discharge, Detect, & Destroy
Wetware
Summary Part
System #1
BBk
ColE2 operon
BBk_131000
Yes
ColE2 w/o pSOS
BBk_131009
Yes
AHL inducible ColE2 with GFP
BBk_131010
Functional test
AHL inducible GFP w/o term. (intermediate)
BBk_131026
Functional test
Part
System #2
Sequenced
BBk
Sequenced
LuxPQ, V. harveyi
BBk_131015
Yes*
LuxOU, V. harveyi
BBk_131016
Yes
qrr4 (quorum-reg. RNA-4) promoter, V. harveyi
BBk_131017
Yes
LuxO inducible RFP
BBk_131019
Functional test
LuxO D47E, V. harveyi
BBk_131022
Yes
LuxO D47A, V. harveyi
BBk_131023
Yes
*Silent mutations to remove EcoRI site in LuxP, EcoRI site in LuxQ, XbaI site in LuxQ
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Conclusions and Summary
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Conclusions and Summary
•
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Establishment of Quorum-coupled Bacteriocin Release Proof-of-Principle: System #1 (AHL-induced ColE2 release)
Discharge, Detect, & Destroy
Wetware
Conclusions and Summary
•
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Establishment of Quorum-coupled Bacteriocin Release Proof-of-Principle: System #1 (AHL-induced ColE2 release)
Discharge, Detect, & Destroy
Wetware
Conclusions and Summary
•
Establishment of Quorum-coupled Bacteriocin Release Proof-of-Principle: System #1 (AHL-induced ColE2 release)
•
Elements required for construction of V. harveyi AI-2 signaling within E. coli deposited in Registry
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Significance
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Significance
•
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Quorum-coupled Bacteriocin Release allows for detection and annihilation of specific Bad Guys based on pheromonal identity
Discharge, Detect, & Destroy
Wetware
Significance
•
Quorum-coupled Bacteriocin Release allows for detection and annihilation of specific Bad Guys based on pheromonal identity
•
Incorporation of Reporter activity allows user to rapidly identify Bad Guy present
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Significance
•
Quorum-coupled Bacteriocin Release allows for detection and annihilation of specific Bad Guys based on pheromonal identity
•
Incorporation of Reporter activity allows user to rapidly identify Bad Guy present
•
AI-2 signaling used by hundreds of bacterial species to coordinate activity within and outside species
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Significance
•
Quorum-coupled Bacteriocin Release allows for detection and annihilation of specific Bad Guys based on pheromonal identity
•
Incorporation of Reporter activity allows user to rapidly identify Bad Guy present
•
AI-2 signaling used by hundreds of bacterial species to coordinate activity within and outside species
•
Quorum Signaling: A new target for antibacterial therapeutics
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Future Directions/Continuing Projects
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Future Directions/Continuing Projects
•
Completion of V. harveyi AI-2 Signaling circuit within E. coli
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Future Directions/Continuing Projects
• •
Completion of V. harveyi AI-2 Signaling circuit within E. coli Selection of species-specific pheromones of pathogenic bacteria (e.g. Cholera Autoinducer-1, CAI-1)
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Future Directions/Continuing Projects
• •
Completion of V. harveyi AI-2 Signaling circuit within E. coli
•
Use of additional bacteriocins (e.g. vibriocins and pyocins)
Selection of species-specific pheromones of pathogenic bacteria (e.g. Cholera Autoinducer-1, CAI-1)
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Acknowledgments
Sonja Georgijevic
Christian Jacob
Bonnie Bassler
Michael Surette
University of Calgary
University of Calgary
Princeton University
University of Calgary
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
Questions
d
Ba
1 # y u G
d Ba y#
Gu 2
Cham p
ion
UNIVERSITY OF
CALGARY
Sunday, April 19, 2009
Discharge, Detect, & Destroy
Wetware
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