iGEM Chiba : E.coli time manager2008.igem.org/files/poster/Chiba.pdf · 1)+AiiA 2)-AiiA 3)No...
1
1)+AiiA 2)-AiiA 3)No Sender ) U A ( e c n e c s e r o u l F P F G Time (min.) iGEM Chiba : E.coli time manager Many electronic devices we use in our daily lives have the ability to keep track of time. For example, a VCR is able to record a TV program at a pre-set time, and a microwave automatically stops heating after a set amount of time. Using these temporal pre-programming functions, we have been liberated from either staying up late to watch a European soccer game or from worrying about our popcorn being burned black while yelling and shouting to the match we have videotaped. In this way, the timer function has revolutionized our lifestyle. 1. Sender cells in a incubator slowly generate signal molecules at a constant rate. The signal molecules are non-degradable (or virtually so in a reasonable time scale) so they get accumulated (linearly) over time in the incubator. 2. Receivers take in the signal molecules and then activate the genetic switch only when the signal concentration reaches a threshold determine inside of the receivers. 3. Either by changing the receiver sensitivity (a) or signal accumulation rate (b), one can freely control the delay interval of the individual switches. Using switches with various times-of-delay, one can sequentially activate many different cellular functions. Time Signal morelecure Gene Expression . c n o c L H A . c n o c L H A Response Time Delay Threshold Gene Expression . c n o c L H A Delay . c n o c L H A Response Time Threshold (a) Producing time delay changing the receiver sensitivity. (b) Producing time delay changing rate of signal accumulation. We thought the same applies to the biotechnology; we want to freely implement such 'timer switches' to various biological functions, preferably both independently and in parallel format. These functions include sensors, synthesizers, or degraders of bioactive compounds/ materials, transportation and secretion machineries, communications, getting/ sticking together, proliferation and cell death. What is E.coli time manager? Signal morelecure Remove AHL Mix Senders (stationaly) Receivers (stationaly) 30 t=0 Measure Fluorescence intensity (t=t 1 ) : Experiment method Measure Fluorescence intensity (t=t 2 ) AHL Receivers cell strain: BW constitutively generating luxR-family protein Plux-gfp constitutively generating luxI-family protein cell strain: XL10Gold AHL Senders Time Tuning the expression level of components Partial quenching of signals LuxR Mutants (Ongoing) Summary Tuning system components Receiver 1) 2,3) Sender S S R R Receiver Sender luxI pTet pTet luxI Two LuxR mutants created by SDM. L42S (1/1000 in sensitivity) (3) construction completed L42A (1/15 in sensitivity) (3) construction completed 1) luxR (high) - Plux-GFP (high) 2) luxR (mid) - Plux-GFP (mid) ) U A ( e c n e c s e r o u l F P F G Time (min.) 0 100 200 300 0 200 400 600 800 1000 1200 1400 1) luxR (high) - Plux-GFP (high) 3) luxR (mid) - Plux-GFP (high) No Sender ) U A ( e c n e c s e r o u l F P F G ) U A ( e c n e c s e r o u l F P F G Time (min.) Communication with inter-species language Speaking to Foreigners Spoken to by Foreigners LuxI from Vibrio fischeri 3OC6HSL O N H O O O RhlI from Pseudomonas aeruginosa C4HSL C6HSL O N H O O O N H O O LasI from Pseudomonas aeruginosa 3OC12HSL O N H O O O Metamorphosizing image (LuxI - LuxR, LasI - LuxR) 0 200 300 400 500 600 0 120 240 360 480 ] . U . A [ y t i s n e t n i e c n e c s e r o u l F Time [min.] ) U A ( e c n e c s e r o u l F P F G ? ? ! ! Hello! ! ? ? @ Hullo? ! Time Delay Test (1) Wang et al.: J. Biol. Chem.279(14),13645-13651,2004. (2)Koch B et.al.: Microbiol. 2005 Nov;151(Pt 11):3589-602. (3)M.K Winson et al.: FEMS Microbiology Letters 163 (1998) 185-192 (4)BBa_F2620:Specificity References System Design using Quorum Sensing luxR pTet GFP pLux luxR pTet GFP pLux aiiA pLac 1) 2) 3) pMB1 ori(high copy) luxR pTet GFP pLux p15A ori(mid copy) luxR pTet GFP pLux pMB1(high copy) p15A(mid copy) luxR pTet GFP pLux Idea: Decreasing the effective concentration of AHL in Receiver cell by expressing AiiA, the enzyme degrades AHL. In general, we need more time in communicating in foreign language than to do so in our native lan- guage. We thought it also applies to the bacterial communications: cross-talking among AHL machin- eries requires higher concentration of signaling molecules than native pairs of senders/ receivers so that it results in significant delay in switching time. Result: The voice (Lux) didn't reach either to American (Las) or to the dog (Rhl). Seems Japanese is not proper as common language... Result: Yes! Japanese (Lux) could understand both language. Interestingly, he instantly understood the language (Rhl) of the dog, while he took a while to understand English(Las). Result: Only the well containing Lux sender/ Lux receiver glowed at 24h. At 48h, the well containing Las sender/ Lux receiver also glowed. Idea: Using Japanese (Lux) as international language. It should properly delay the commu- nication! Idea: Vice versa. Japanese trying to understand the voice of American (Las) and of the dog (Rhl). The communication should delay. Result: AiiA was too active ... Almost no communication observed. Idea: Makin “dull” receivers by changing the ratio of luxR & its target(plux) 1. Using 3OC12HSL (Las-type) instead of 3OC6HSL (Lux-type) as signal molecule, we could realize an available delayed-switch of LuxR/LuxP. The delay interval was 2 hours, in which we confirmed the GFP expression by visual judgement. 2. We demonstrated a drawing by means of the sender-receiver mixture of LasI-LuxR and LuxI-LuxR as E.coli paints. 3. Interestingly, we kept failing to observe the crosstalk between Lux-sender and receivers from other organisms. 4. Several other strategies have been tested, too. We have positive expectation especially on the engineering of LuxR protein. Idea: Changing the binding constant of luxR to AHL Result: Yet to be seen, it should work! S R Receiver L42S/A dull mutant Sender pTet luxI 1) luxR pTet GFP pLux S R rhll pLac lasI pLac 100 200 300 400 0 200 400 600 800 1000 1200 1400 GFP Fl uor es cence ( A. U. ) Ti me[m i n. ] pTet GFP pLux LasR RhlR LuxR Lux Lux Lux Las Lux Rhl luxI pLac luxR pTet GFP pLux Rhl Lux Las Lux Visiblezone Lux Lux
Transcript of iGEM Chiba : E.coli time manager2008.igem.org/files/poster/Chiba.pdf · 1)+AiiA 2)-AiiA 3)No...
1)+AiiA
2)-AiiA
3)No Sender
)U
A( ecnecseroulF PFG
Time (min.)
iGEM Chiba : E.coli time managerMany electronic devices we use in our daily lives have the ability to keep track of time. For example, a VCR is able to record a TV program at a pre-set time, and a microwave automatically stops heating after a set amount of time. Using these temporal pre-programming functions, we have been liberated from either staying up late to watch a European soccer game or from worrying about our popcorn being burned black while yelling and shouting to the match we have videotaped. In this way, the timer function has revolutionized our lifestyle.
1. Sender cells in a incubator slowly generate signal molecules at a constant rate. The signal molecules are non-degradable (or virtually so in a reasonable time scale) so they get accumulated (linearly) over time in the incubator.
2. Receivers take in the signal molecules and then activate the genetic switch only when the signal concentration reaches a threshold determine inside of the receivers.
3. Either by changing the receiver sensitivity (a) or signal accumulation rate (b), one can freely control the delay interval of the individual switches. Using switches with various times-of-delay, one can sequentially activate many different cellular functions.
Time
Signalmorelecure
Gene Expression
.cnoc LHA
.cnoc LHA
Response Time
Delay
Threshold
Gene Expression
.cnoc LHADelay
.cnoc LHA
Response Time
Threshold
(a) Producing time delay changing the receiver sensitivity.
(b) Producing time delay changing rate of signal accumulation.
We thought the same applies to the biotechnology; we want to freely implement such 'timer switches' to various biological functions, preferably both independently and in parallel format. These functions include sensors, synthesizers, or degraders of bioactive compounds/ materials, transportation and secretion machineries, communications, getting/ sticking together, proliferation and cell death.
What is E.coli time manager?
Signalmorelecure
RemoveAHL
Mix
Senders(stationaly)
Receivers(stationaly)
30℃t=0
MeasureFluorescence
intensity
(t=t1)
: Experiment method
MeasureFluorescence
intensity
(t=t2)
AHL Receivers cell strain: BWconstitutively generating luxR-family proteinPlux-gfp
constitutively generating luxI-family proteincell strain: XL10GoldAHL Senders Time
Tuning the expression level of components
Partial quenching of signals
LuxR Mutants (Ongoing)
Summary
Tuning system components
Receiver
1)
2,3)
Sender
S
S
R
R
ReceiverSender
HighluxIpTet
luxIpTet
pTet luxI
High/MidluxRpTet
High/MidGFPpLux
Two LuxR mutants created by SDM. L42S (1/1000 in sensitivity)(3) ：construction completed L42A (1/15 in sensitivity)(3)：construction completed
1) luxR (high) - Plux-GFP (high)
2) luxR (mid) - Plux-GFP (mid)
)U
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Time (min.)
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A ( yt isn et ni ecn ecs er oulF
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No Sender
)UA( ecnecseroulF PFG
)U
A( e cnec seroulF PFG
Time (min.)
Communication with inter-species language
Speaking to Foreigners
Spoken to by Foreigners
Intensity reached at 200 was 2.5 hours longer. The increase of fluorescence intensity caused by 3OC12HSL was more gradualy than that caused by 3OC6HSL
LuxI from Vibrio fischeri
3OC6HSL
ONH
OO
O
RhlI from Pseudomonas aeruginosa
C4HSL C6HSL
ONH
O
O
ONH
O
O
LasI from Pseudomonas aeruginosa
3OC12HSL
ONH
OO
O
?
Metamorphosizing image (LuxI - LuxR, LasI - LuxR)
luxIpLacHigh
lasIpLacHigh
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].U.
A[ytisnetni ecnecseroulF
Time [min.]
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A( ecnecseroulF PFG
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0 120 240 360 480
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A[ytisnetni ecnecseroulF
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GFP Fluorescence : 200Lower limit which can be confirmed GFP expression by viewing.
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Receivers
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No Sender
Sendersand
Receivers
)U
A ( ecn ecse ro ulF PFG
?
?
コンニチハ!
コンニチハ!
Hello!
コンニチハ!
?
?＃ё@♀！
Hullo?
コンニチハ!
Time Delay Test
(1) Wang et al.: J. Biol. Chem.279(14),13645-13651,2004.(2)Koch B et.al.: Microbiol. 2005 Nov;151(Pt 11):3589-602. (3)M.K Winson et al.: FEMS Microbiology Letters 163 (1998) 185-192(4)BBa_F2620:Specificity
References
System Design using Quorum Sensing
luxRpTet GFPpLux
luxRpTet GFPpLux
aiiApLac
1)
2)
3)
pMB1 ori(high copy)luxRpTet GFPpLux
p15A ori(mid copy)luxRpTet GFPpLux
pMB1(high copy) p15A(mid copy)luxRpTet GFPpLux
Idea: Decreasing the effective concentration of AHL in Receiver cell by expressing AiiA, the enzyme degrades AHL.
In general, we need more time in communicating in foreign language than to do so in our native lan-guage. We thought it also applies to the bacterial communications: cross-talking among AHL machin-eries requires higher concentration of signaling molecules than native pairs of senders/ receivers so that it results in significant delay in switching time.
Result: The voice (Lux) didn't reach either to American (Las) or to the dog (Rhl). Seems Japanese is not proper as common language...
Result: Yes! Japanese (Lux) could understand both language. Interestingly, he instantly understood the language (Rhl) of the dog, while he took a while to understand English(Las).
Result: Only the well containing Lux sender/ Lux receiver glowed at 24h. At 48h, the well containing Las sender/ Lux receiver also glowed.
Idea: Using Japanese (Lux) as international language. It should properly delay the commu-nication!
Idea: Vice versa. Japanese trying to understand the voice of American (Las) and of the dog (Rhl). The communication should delay.
Result: AiiA was too active ... Almost no communication observed.
Idea: Makin “dull” receivers by changing the ratio of luxR & its target(plux)
1. Using 3OC12HSL (Las-type) instead of 3OC6HSL (Lux-type) as signal molecule, we could realize an available delayed-switch of LuxR/LuxP. The delay interval was 2 hours, in which we confirmed the GFP expression by visual judgement.2. We demonstrated a drawing by means of the sender-receiver mixture of LasI-LuxR and LuxI-LuxR as E.coli paints.
3. Interestingly, we kept failing to observe the crosstalk between Lux-sender and receivers from other organisms.
4. Several other strategies have been tested, too. We have positive expectation especially on the engineering of LuxR protein.
Idea: Changing the binding constant of luxR to AHL
Result: Yet to be seen, it should work!
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150
200
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Receivers
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G)
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UA ( ecn ecse ro ulF PF
G)
GFPpLux
rhlRpTet
?S R
?
GFPpLux
lasRpTet
S R
S R
luxIpLacS Sender is all same in these experiments!
2.RhlR 3.LasR
S R
Receiver
L42S/A
dull mutant
Sender
pTet luxI1)
luxRpTet GFPpLux
S R
rhllpLac
lasIpLac
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ence
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Ti me[ mi n. ]
pTet GFPpLux
LasR
RhlR
LuxR
Lux Lux
Lux LasLux Rhl
luxIpLac
luxRpTet GFPpLux
Rhl Lux
Las Lux
Visible zone
Lux Lux
