iGEM 2008 Calgary Wetware Full)

Quorum-coupled Bacteriocin Release:
Engineering a Champion
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AI
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Thane Kubik
☠
Kevin McLeod
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Dave Curran
Kingsley Nze
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CALGARY Discharge, Detect, & Destroy Wetware
Sunday, April 19, 2009
Discharge, Detect, & Destroy
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# 1
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What is quorum sensing?
Microbial Talk
nerd
chatter to m y
e!
c t e r i a t a l k
H ow ba
c h o t h e r
to ea
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Quorum sensing: establishing a consensus
Number of cells (log)

Time
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Time
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Time
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Time
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Time
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Time
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Do bacteria have anything interesting to say, Doctor??
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Talk of virulence
• Vibrio cholerae
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Talk of virulence
• Vibrio cholerae
Decisions of biofilm formation

• Pseudomonas aeruginosa
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Talk of virulence
• Vibrio cholerae

Dialogue of genetic competence

• Streptococcus pneumoniae
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Talk of virulence
• Vibrio cholerae

Dialogue of genetic competence

• Streptococcus pneumoniae
Chattering with human host

• Enterococcus faecalis
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V. harveyi - sending mixed messages
Two Quorum Sensing systems
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AHL
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AHL
Intimate
conversations
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AHL AI-2
Intimate
conversations
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AHL AI-2
Intimate Communal
conversations living
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System #1: Champion vs. Bad Guy#1
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Autoinducer-1 (AI-1/AHL) Signaling in V. harveyi
er
nd
Se
Receiv
er
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LuxR
er
nd
Se
Receiv
er
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L
AH
LuxR
er
nd
Se
Receiv
er
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L
AH
LuxR
er
nd
Se
Receiv
er
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er
L
nd
AH
LuxR
Se
Receiv
er
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er
L
nd
AH
LuxR
Se
Receiv
er
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Champion vs. Bad Guy#1
Champ
ion
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LuxR
Champ
ion
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LuxR
y#
Gu
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Ba
Champ
ion
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L
AH
1
LuxR
y#
Gu
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Ba
Champ
ion
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L
AH
1
LuxR
y#
Gu
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Champ
ion
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1
y#
Gu
L
AH
LuxR
d
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Champ
ion
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1
y#
Gu
L
AH
LuxR
d
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Champ
ion
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1
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Gu
L
AH
LuxR
d
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C
I
L
Champ
ion
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L
AH
LuxR
C
I
L
Champ
ion
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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
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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) 2 ColE2 in BL21 + MytC (2hr)
3 ColE2 in BL21 + MytC (4hr) 3 ColE2 in BL21 + MytC (4hr)
4 LB + MytC 4 LB + MytC
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Can Bad Guy #1 discharge AHL? Can Champion detect?
# Spot
1 2 1 Bad Guy #1
3 4 2 AHL
3 AHL
4 Bad Guy #1
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Can Champion detect and destroy?
• Does Bad Guy #1 induce Champion to

express GFP and release ColE2?
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Can Champion detect and destroy?
• Does Bad Guy #1 induce Champion to

express GFP and release ColE2?
• Concentration-dependent response?
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Champ
Champ
Champ
Champ
Champ
Champ
Champ
Champ
1 2 3 4 5 6 7 8
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Time
Champ
Champ
Champ
Champ
Champ
Champ
Champ
Champ
1 2 3 4 5 6 7 8
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AHL
Time 0.5 µM 0.75 µM 1.25 µM 2.5 µM
Champ
Champ
Champ
Champ
Champ
Champ
Champ
Champ
1 2 3 4 5 6 7 8
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AHL BG#1
Time 0.5 µM 0.75 µM 1.25 µM 2.5 µM 1/30 1/15 1/6 1/3
Champ
Champ
Champ
Champ
Champ
Champ
Champ
Champ
Champ
Champ
Champ
Champ
1 2 3 4 5 6 7 8
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AHL BG#1
0.5 µM 0.75 µM 1.25 µM 2.5 µM 1/30 1/15 1/6 1/3
Champ
Champ
Champ
Champ
Champ
Champ
Champ
Champ
Champ
Champ
Champ
Champ
1 2 3 4 5 6 7 8
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AHL BG#1
0.5 µM 0.75 µM 1.25 µM 2.5 µM 1/30 1/15 1/6 1/3
Champ
Champ
Champ
Champ
Champ
Champ
Champ
Champ
Champ
Champ
Champ
Champ
1 2 3 4 5 6 7 8
Samples taken:
1 hour post induction
2 hours post induction
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AHL BG#1
0.5 µM 0.75 µM 1.25 µM 2.5 µM 1/30 1/15 1/6 1/3
Champ
Champ
Champ
Champ
Champ
Champ
Champ
Champ
Champ
Champ
Champ
Champ
1 2 3 4 5 6 7 8
Samples taken:
1 hour post induction
lawn of BG#1
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AHL BG#1
0.5 µM 0.75 µM 1.25 µM 2.5 µM 1/30 1/15 1/6 1/3
Champ
Champ
Champ
Champ
Champ
Champ
Champ
Champ
Champ
Champ
Champ
Champ
1 2 3 4 5 6 7 8
Samples taken:
1 hour post induction 1 hr 2 hr
4 hours post induction 4 hr C
lawn of BG#1
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... and the Champion prevails
0.5 µM AHL 0.75 µM AHL
1.25 µM AHL 2.5 µM AHL
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... 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
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Our Champion in action
BG#1/CC = 1/30 BG#1/CC = 1/30
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System #2:
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LuxP
LuxQ
LuxU
LuxR mRNA
LuxO
Receiver
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LuxP
LuxQ
LuxU P
LuxU
LuxR mRNA
LuxO
Receiver
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LuxP
LuxQ
LuxU P
LuxU
LuxR mRNA
LuxO P
LuxO
Receiver
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LuxP
LuxQ
LuxU P
LuxU
P LuxR mRNA
LuxO
LuxO P
LuxO
Receiver
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LuxP
LuxQ
LuxU P
LuxU
P LuxR mRNA
LuxO 54
σ
LuxO P
LuxO
Receiver
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LuxP
LuxQ
LuxU P
LuxU
LuxR mRNA
LuxO P
LuxO
P
LuxO 54
σ
Receiver
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LuxP
LuxQ
LuxU P
LuxU
LuxR mRNA
LuxO P
LuxO
P sRNA
LuxO 54
σ
Receiver
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LuxP
LuxQ
LuxU P
LuxU
LuxR mRNA
LuxO P
LuxO
P sRNA
LuxO 54
σ
Receiver
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LuxP
LuxQ
LuxU P
LuxU
LuxR mRNA
LuxO P
LuxO
P sRNA
LuxO 54
σ
Receiver
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AI-2 AI
-2 AI-2
AI-2
AI-2
AI-2
AI-2 2
AI-
LuxP
AI-2 LuxQ
LuxU P
LuxU
LuxR mRNA
AI-2
LuxO P
LuxO
P sRNA
LuxO 54
σ
AI-2
Receiver
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AI-2 AI
-2 AI-2
AI-2
AI-2
AI-2 2
AI-
AI-2
LuxP
AI-2 LuxQ
LuxU P
LuxU
LuxR mRNA
AI-2
LuxO P
LuxO
P sRNA
LuxO 54
σ
AI-2
Receiver
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AI-2 AI
-2 AI-2
AI-2
AI-2
AI-2 2
AI-
AI-2
LuxP
AI-2 LuxQ
LuxU P
LuxU
P
LuxR mRNA
AI-2
LuxO P
LuxO
P sRNA
LuxO 54
σ
AI-2
Receiver
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AI-2 AI
-2 AI-2
AI-2
AI-2
AI-2 2
AI-
AI-2
LuxP
AI-2 LuxQ
LuxU P
LuxU
P
LuxR mRNA
AI-2
LuxO P
LuxO
P sRNA
LuxO 54
σ
P
AI-2
Receiver
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AI-2 AI
-2 AI-2
AI-2
AI-2
AI-2 2
AI-
AI-2
LuxP
AI-2 LuxQ
LuxU P
LuxU
P
LuxR mRNA
AI-2
LuxO P
LuxO
sRNA
AI-2
Receiver
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AI-2 AI
-2 AI-2
AI-2
AI-2
AI-2 2
AI-
AI-2
LuxP
AI-2 LuxQ
LuxU P
LuxU
P
LuxR mRNA
AI-2
LuxO P
LuxO
sRNA
AI-2
Receiver
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AI-2 AI
-2 AI-2
AI-2
AI-2
AI-2 2
AI-
AI-2
LuxP
AI-2 LuxQ
LuxU P
LuxU
P
LuxR mRNA
AI-2
LuxO P
LuxO
AI-2
Receiver
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AI-2 AI
-2 AI-2
AI-2
AI-2
AI-2 2
AI-
AI-2
LuxP
AI-2 LuxQ
LuxU P
LuxU
P
LuxR mRNA
AI-2
LuxO P LuxR
LuxO
AI-2
Receiver
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AI-2 AI
-2 AI-2
AI-2
AI-2
AI-2 2
AI-
AI-2
LuxP
AI-2 LuxQ
LuxU P
LuxU
P
LuxR mRNA
AI-2
LuxO P
LuxO
LuxR
AI-2
Receiver
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AI-2 AI
-2 AI-2
AI-2
AI-2
AI-2 2
AI-
AI-2
LuxP
AI-2 LuxQ
LuxU P
LuxU
P
LuxR mRNA
AI-2
LuxO P
LuxO
LuxR
AI-2
Receiver
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Why is AI-2 signaling worth all of the blood, sweat and tears??
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Over 50% bacteria (i.e. >300 species) whose genome has been
sequenced encode LuxS (required for AI-2 production)
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Genes controlled by AI-2 signaling:

•E. coli OI57:H7: Virulence, motility, secretion
•Streptococcus pneumoniae: Competence,Virulence, Persistence
•Vibrio cholera: Virulence, Biofilms, Toxins
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Addition of a second signaling system to the Registry
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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)
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LuxP
LuxQ
LuxU
LuxO
Champion
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LuxP
LuxQ
LuxU P
LuxU
LuxO
Champion
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LuxP
LuxQ
LuxU P
LuxU
LuxO P
LuxO
Champion
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LuxP
LuxQ
LuxU P
LuxU
P
LuxO
LuxO P
LuxO
Champion
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LuxP
LuxQ
LuxU P
LuxU
P
LuxO 54
σ
LuxO P
LuxO
Champion
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LuxP
LuxQ
LuxU P
LuxU
LuxO P
LuxO
P
LuxOσ54
Champion
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LuxP
LuxQ
LuxU P
LuxU
LuxO P
LuxO
P R
LuxOσ54
Champion
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LuxP
LuxQ
LuxU P
LuxU
LuxO P
LuxO
P
LuxOσ54 R
Champion
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LuxP
LuxQ
LuxU P
LuxU
LuxO P
LuxO
P
LuxOσ54 R
Champion
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Bad Guy #2
LuxP
LuxQ
LuxU P
LuxU
LuxO P
LuxO
P
LuxOσ54 R
Champion
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AI-2
AI
-2 Bad Guy #2
AI-2
AI-2
2
AI-
LuxP
LuxQ
AI-2
AI-2
LuxU P
LuxU
AI-2
LuxO P
LuxO
P
LuxOσ54 R
Champion
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AI
-2 Bad Guy #2
AI-2
AI-2
2
AI-
AI-2
LuxP
LuxQ
AI-2
AI-2
LuxU P
LuxU
AI-2
LuxO P
LuxO
P
LuxOσ54 R
Champion
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AI
-2 Bad Guy #2
AI-2
AI-2
2
AI-
AI-2
LuxP
LuxQ
AI-2
AI-2
LuxU P
LuxU
AI-2
P
LuxO P
LuxO
P
LuxOσ54 R
Champion
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AI
-2 Bad Guy #2
AI-2
AI-2
2
AI-
AI-2
LuxP
LuxQ
AI-2
AI-2
LuxU P
LuxU
AI-2
P
LuxO P
LuxO
P P
LuxOσ54 R
Champion
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AI
-2 Bad Guy #2
AI-2
AI-2
2
AI-
AI-2
LuxP
LuxQ
AI-2
AI-2
LuxU P
LuxU
AI-2
P
LuxO P
LuxO
P
R
Champion
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AI
-2 Bad Guy #2
AI-2
AI-2
2
AI-
AI-2
LuxP
LuxQ
AI-2
AI-2
LuxU P
LuxU
AI-2
P
LuxO P
LuxO
P
R
Champion
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AI
-2 Bad Guy #2
AI-2
AI-2
2
AI-
AI-2
LuxP
LuxQ
AI-2
AI-2
LuxU P
LuxU
AI-2
P
LuxO P
LuxO
Champion
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AI
-2 Bad Guy #2
AI-2
AI-2
2
AI-
AI-2
LuxP
LuxQ
AI-2
AI-2
LuxU P
LuxU
AI-2
P
LuxO P
LuxO
Champion
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AI
-2 Bad Guy #2
AI-2
AI-2
2
AI-
AI-2
LuxP
LuxQ
AI-2
AI-2
LuxU P
LuxU
AI-2
P
LuxO P
LuxO
P C I L
Champion
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AI
-2 Bad Guy #2
AI-2
AI-2
2
AI- C
AI-2
LuxP
LuxQ
AI-2
AI-2
LuxU P
LuxU
AI-2
P
LuxO P
LuxO
P C I L
Champion
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AI
-2
AI-2
AI-2
2
AI-
AI-2
LuxP
LuxQ
AI-2
AI-2
LuxU P
LuxU
AI-2
P
LuxO P
LuxO
P C I L
Champion
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Engineering the Champion
AI-2
LuxP
LuxQ
LuxU P
Signaling
LuxU
LuxO P
LuxO
P Response
Champion
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Signaling circuit
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Signaling circuit
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Signaling circuit
PCR amplification
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Signaling circuit
PCR amplification
TOPO Cloning
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Signaling circuit
PCR amplification
TOPO Cloning
Verification by Colony PCR/

Restriction Digest/Sequencing
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Signaling circuit
PCR amplification
TOPO Cloning

PCR amplification
with BioBrick primers
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Signaling circuit
PCR amplification
TOPO Cloning

PCR amplification
BioBrick Cloning and

Circuit(s) assembly
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Signaling circuit
PCR amplification
TOPO Cloning

Mutagenesis
PCR amplification
BioBrick Cloning and

Circuit(s) assembly
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Response circuit
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Testing the Response circuit
LuxO D47E LuxO D47A

mimics LuxO-℗ = active inactive
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LuxO D47E LuxO D47A

P
LuxO
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LuxO D47E LuxO D47A

P
LuxO
σ54
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LuxO D47E LuxO D47A

P
LuxO 54
σ
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LuxO D47E LuxO D47A

P
LuxO 54
σ R
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LuxO D47E LuxO D47A

P
LuxO 54 R
σ R
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LuxO D47E LuxO D47A

P
LuxO 54 R
σ R
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LuxO D47E LuxO D47A

P
LuxO 54 R
σ R
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LuxO D47E LuxO D47A

P
LuxO 54 R
σ R
NO RFP, NO ColE3
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LuxO D47E LuxO D47A

LuxO
P
LuxO 54 R
σ R
NO RFP, NO ColE3
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LuxO D47E LuxO D47A

σ54
LuxO
P
LuxO 54 R
σ R
NO RFP, NO ColE3
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LuxO D47E LuxO D47A

LuxO
P
LuxO 54 R
σ R
NO RFP, NO ColE3
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LuxO D47E LuxO D47A

LuxO
P
LuxO 54 R
σ R
NO RFP, NO ColE3
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LuxO D47E LuxO D47A

LuxO
P
LuxO 54 R
σ R
NO RFP, NO ColE3 RFP & ColE3
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Summary
Part BBk Sequenced
ColE2 operon BBk_131000 Yes
ColE2 w/o pSOS BBk_131009 Yes

System #1 AHL inducible ColE2 with GFP BBk_131010
Functional
test
Functional
AHL inducible GFP w/o term. (intermediate) BBk_131026 test
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Summary
Part BBk Sequenced
ColE2 operon BBk_131000 Yes
ColE2 w/o pSOS BBk_131009 Yes

System #1 AHL inducible ColE2 with GFP BBk_131010
Functional
test
Functional
AHL inducible GFP w/o term. (intermediate) BBk_131026 test
Part 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

System #2 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
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Conclusions and Summary
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• Establishment of Quorum-coupled Bacteriocin

Release Proof-of-Principle: System #1 (AHL-induced
ColE2 release)
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ColE2 release)
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ColE2 release)
• Elements required for construction of V. harveyi AI-2

signaling within E. coli deposited in Registry
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Significance
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Significance
• Quorum-coupled Bacteriocin Release allows for

detection and annihilation of specific Bad Guys based on
pheromonal identity
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Significance

pheromonal identity
• Incorporation of Reporter activity allows user to rapidly

identify Bad Guy present
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Significance

pheromonal identity

• AI-2 signaling used by hundreds of bacterial species to

coordinate activity within and outside species
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Significance

pheromonal identity

• 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
Future Directions/Continuing Projects
UNIVERSITY OF
• Completion of V. harveyi AI-2 Signaling circuit within E. coli
UNIVERSITY OF
• Selection of species-specific pheromones of pathogenic

bacteria (e.g. Cholera Autoinducer-1, CAI-1)
UNIVERSITY OF
• Selection of species-specific pheromones of pathogenic

bacteria (e.g. Cholera Autoinducer-1, CAI-1)
• Use of additional bacteriocins (e.g. vibriocins and pyocins)
UNIVERSITY OF
Acknowledgments
Sonja Georgijevic Christian Jacob Bonnie Bassler Michael Surette

University of Calgary University of Calgary Princeton University University of Calgary
UNIVERSITY OF
Questions
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UNIVERSITY OF

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Quorum-coupled Bacteriocin Release:

Number of cells (log)

Number of cells (log)

Number of cells (log)

Number of cells (log)

Number of cells (log)

Number of cells (log)

Decisions of biofilm formation

Decisions of biofilm formation

Dialogue of genetic competence

Decisions of biofilm formation

Dialogue of genetic competence

Chattering with human host

• Does Bad Guy #1 induce Champion to

• Does Bad Guy #1 induce Champion to

0.5 µM AHL 0.75 µM AHL

1.25 µM AHL 2.5 µM AHL

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

BG#1/CC = 1/30 BG#1/CC = 1/30

Genes controlled by AI-2 signaling:

Genes controlled by AI-2 signaling:

Genes controlled by AI-2 signaling:

Verification by Colony PCR/

Verification by Colony PCR/

Verification by Colony PCR/

BioBrick Cloning and

Verification by Colony PCR/

BioBrick Cloning and

LuxO D47E LuxO D47A

LuxO D47E LuxO D47A

LuxO D47E LuxO D47A

LuxO D47E LuxO D47A

LuxO D47E LuxO D47A

LuxO D47E LuxO D47A

LuxO D47E LuxO D47A

LuxO D47E LuxO D47A

LuxO D47E LuxO D47A

LuxO D47E LuxO D47A

LuxO D47E LuxO D47A

LuxO D47E LuxO D47A

LuxO D47E LuxO D47A

LuxO D47E LuxO D47A

NO RFP, NO ColE3 RFP & ColE3

ColE2 w/o pSOS BBk_131009 Yes

ColE2 w/o pSOS BBk_131009 Yes

Part BBk Sequenced

LuxOU, V. harveyi BBk_131016 Yes

qrr4 (quorum-reg. RNA-4) promoter, V. harveyi BBk_131017 Yes

LuxO D47E, V. harveyi BBk_131022 Yes

LuxO D47A, V. harveyi BBk_131023 Yes

• Establishment of Quorum-coupled Bacteriocin

• Establishment of Quorum-coupled Bacteriocin

• Establishment of Quorum-coupled Bacteriocin

• Elements required for construction of V. harveyi AI-2

• Quorum-coupled Bacteriocin Release allows for

• Quorum-coupled Bacteriocin Release allows for

• Incorporation of Reporter activity allows user to rapidly

• Quorum-coupled Bacteriocin Release allows for

• Incorporation of Reporter activity allows user to rapidly

• AI-2 signaling used by hundreds of bacterial species to

• Quorum-coupled Bacteriocin Release allows for

• Incorporation of Reporter activity allows user to rapidly

• AI-2 signaling used by hundreds of bacterial species to