Team:Chiba/Project/Experiments:Sender Crosstalk

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==Sender Crosstalk==
==Sender Crosstalk==
=== Design===
=== Design===
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[[Image:AHL variety chiba.gif|frame|left|'''Fig. 2　AHL varieties''']]
[[Image:AHL variety chiba.gif|frame|left|'''Fig. 2　AHL varieties''']]
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Each species has their own LuxI-type proteins,which synthesize their specific autoinducers, AHLs. AHLs produced by different LuxI-type proteins differ only in the length of the acyl-chain moiety and substitution at position C-3.LuxR,which is original for Vibrio fischeri, is activated by its cognate autoinducer, 3OC6HSL. However, LuxR is also activated by non-endogenous molecules, C4HSL, C6HSL, and 3OC12HSL. Activation by non-endogenous molecules requires a higher signal concentration (2). This results in slower activation of receivers, when AHL concentration is increasing.
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Each species has their own LuxI-type proteins,which synthesize their specific autoinducers, AHLs. AHLs produced by different LuxI-type proteins differ only in the length of the acyl-chain moiety and substitution at position C-3[[Team:Chiba/Project/Experiments:Sender Crosstalk#references|<sup>(4)</sup>]].LuxR,which is original for Vibrio fischeri, is activated by its cognate autoinducer, 3OC6HSL. However, LuxR is also activated by non-endogenous molecules, C4HSL, C6HSL, and 3OC12HSL. Activation by non-endogenous molecules requires a higher signal concentration [[Team:Chiba/Project/Experiments:Sender Crosstalk#references|<sup>(1),(2)</sup>]]. This results in slower activation of receivers, when AHL concentration is increasing.
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To characterize quorum sensing crosstalk, constitutive AHL senders were mixed with constitutive receivers and measure fluorescence intensity.
To characterize quorum sensing crosstalk, constitutive AHL senders were mixed with constitutive receivers and measure fluorescence intensity.
   
   
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#Transformed Senders into ''E. coli'' strains (XL10Gold) and Receiver into ''E. coli'' strain (JW1908).
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#Transformed Senders into ''E. coli'' strains (XL10Gold or JW1908) and Receiver into ''E. coli'' strain (JW1908).
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#Inoculated them independently in liquid media. Incubated at 37°C for 12hours.
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#Inoculated them independently in liquid media. Incubated at 37&deg;C for 12hours.
#Mixed them.
#Mixed them.
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#Incubated at 30&deg;C.
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#Incubated at 37&deg;C or 30&deg;C.
#Measured intensity of green fluorescence (485 nm (excitation) and 527 nm (emission)) at regular time intervals.
#Measured intensity of green fluorescence (485 nm (excitation) and 527 nm (emission)) at regular time intervals.
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===Results & Discussion===
===Results & Discussion===
[[image:senders_crosstalk_chiba_01.gif|frame|
[[image:senders_crosstalk_chiba_01.gif|frame|
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'''Fig. 3　senders crosstalk test.'''senders strain XL10Gold,Receiver strain JW1908.Reaction temparature was 30°C.All measurements are averages from three replicate cultures with error bars representing standard deviations.Labeling:LuxI, RhlI, and LasI means fluorescence induced by AHLs synthesized by LuxI, RhlI, and LasI respectively.]]
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'''Fig. 3　senders crosstalk test.'''senders strain XL10Gold,Receiver strain JW1908.Reaction temparature was 30&deg;C.All measurements are averages from three replicate cultures with error bars representing standard deviations.Labeling:LuxI, RhlI, and LasI means fluorescence induced by AHLs synthesized by [http://partsregistry.org/Part:BBa_K084012 BBa_K084012], [http://partsregistry.org/Part:BBa_K084008 BBa_K084008], and [http://partsregistry.org/Part:BBa_K084007 BBa_K084007] respectively.]]
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====[[Team:Chiba/Sender experiments/Senders(XL10Gold) T9002(JW1908)#Reaction temparature:30°C|Senders(XL10Gold), T9002(JW1908)@30&deg;C, Sender(&mu;L):Receiver(&mu;L)=500：500, 100:1000, 10:1000]]====
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====[[Team:Chiba/Sender experiments/Senders(XL10Gold) T9002(JW1908)#Reaction temparature:30&deg;C|Senders(XL10Gold), T9002(JW1908)@30&deg;C, Sender(&mu;L):Receiver(&mu;L)=500：500, 100:1000, 10:1000]]====
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*LasIを形質転換入した細胞は37&deg;Cよりも、30&deg;Cにおいて盛んにAHLを合成している(と思われる).
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When incubated at 30&deg;C,E.coli strain(XL10Gold) transformed with the LasI genes produced more AHL than at 37&deg;C.
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At 30 degrees Celsius, the ''E. coli'' (XL10G) transformed with the LasI genes composed more AHL than 37 degrees Celsius.
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（intensity of green fluorescence after incubating for 8 hours at 37&deg;C：30&deg;C=163：245）
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*LasI gene(BBa_K084007)によって合成された3OC12HSLとLuxI gene(BBa_K084012)によって合成される3OC6HSLによって合成されるAHLは,ともにLuxRタンパク質を活性化した.3OC12HSLの発現による蛍光強度の上昇は,3OC6HSLのそれよりもゆるやかであり,蛍光強度200に達するまでの時間に2．5時間の差がみられた.その時の条件は,培養液体積比=1:1,培養温度30&deg;C,AHL　senderの株は,XL10Gold,AHL receiverの株はJW1908であった.
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*3OC12HSL synthesized by LasI protein and 3OC6HSL synthesized by LuxI protein both activated LuxR protein and cousese gfp expression .The increase of green fluorescence intensity caused by 3OC12HSL was more gradualy than that caused by 3OC6HSL,time before fluorescence intensity reached at 200 was 2.5 hours longer than that of the 3OC6HSL.
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Experimental conditions were shown in below:
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*reaction temparature was 30&deg;C.
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*The ratio of culture volumes was 1:1.
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*LuxI gene was transformed with ''E. coli'' strain XL10Gold. LasI gene was transformed with ''E. coli'' strain JW1908.
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*The sender genes LuxI and LasI was produce the greatest time difference, 2.5 hours.
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*Rhlのタグなしとタグ付きの差は、８ｈ後の最終強度にしか現れなかった。
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*We observed the difference  between Rhl and Rhl on last data which was measured 8 hours later.
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*LVA-tag had effect only on the maximum fluorescence intensity induced by two signal molecules.  
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:また、差が一番でたのが30°Cで行ったこの実験で、タグなし：タグつき＝507：456。
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(Green fluorescence intensity after incubating for 8 hours was:
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3OC6HSL,3OC12HSLに対する、LuxRの応答時間に、二時間の差ができた（3OC12HSLの場合、3OC6HSLに比べて二時間遅れて応答する）。
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::163 at 37&deg;C.
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(そのときの条件は、培養温度30°C、genelatorの株はXL10Gold,Receiverの株はJW1908のとき、であった。) <??
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::245 at 30&deg;C.
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3OC12HSL produced by LasI protein(expressed by[http://partsregistry.org/Part:BBa_K084007 BBa_K084007] and 3OC6HSL produced by LuxI protein(expressed by[http://partsregistry.org/Part:BBa_K084012 BBa_K0840012]) both activated LuxR protein and cousese gfp expression.The increase of green fluorescence intensity caused by 3OC12HSL was more gradualy than that caused by 3OC6HSL,time before fluorescence intensity reached at 200 was 2.5 hours longer than that of the 3OC6HSL.
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*LasI gene worked more actively at 30&deg;C than worked at 37&deg;C.(Fuluorecence intensity after 8 hours incubating at 37&deg;C was 163,on the other hand,Fuluorecence intensity after 8 hours incubating at 30&deg was 245.)
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*The time to achieve 200 of fuluorecence intensity with LasI and LuxR (pLac)(=1:1) was delayed 2.5 hours.
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*RhlI and RhlI (with LVA tag) made a difference of the latest intensities after 8 hours.
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:And,this conbination (@30&deg;C) made a greatest differences.(no LVA tag):(LVA tag)=507:456.
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When we used the cross-talk strategy using 3OC12HSL instead of 3OC6HSL, the delayed switch worked 2 hours after the original switch worked. Futher variation of delayed switches will be realized:
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===Future plans===
===Future plans===
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*To make the slope of transfer curve of our device to be steep:
*To make the slope of transfer curve of our device to be steep:
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#Introduce Positive Feedback Loop into our gene circuit.
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#Introduce Positive Feedback Loop into our genetic circuit.
===Other Experiments===
===Other Experiments===
====Visual Judgement====
====Visual Judgement====
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*Fluorescence intensity was from 150 to 200 when the 2 mL of reaction culture in a test tube.
 
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====[[Team:Chiba/Sender experiments/Senders(JW1908) T9002(JW1908)#Reaction temparature:37°C|Senders(JW1908), T9002(JW1908)@37°C, Sender(&mu;L):Receiver(&mu;L)=500：500, 100:1000, 10:1000]]====
 
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*Among RhlI, LasI, and CinI+LVA, RhlI+LVA is the easist to cross-talk with LuxR
 
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*Delay time by cross-talk among senders are generated only a range of one hour to amount
 
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to 200 of fluorescence intensity.
 
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====[[Team:Chiba/Sender experiments/Senders(JW1908) T9002(JW1908)#Reaction temparature:30°C|Senders(JW1908),T9002(JW1908)@30°C,Sender(&mu;L):Receiver(&mu;L)=500：500,100:1000,10:1000]]====
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fluorescence intensity was from 150 to 200 when the 2 mL of reaction culture in a test tube.
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====[[Team:Chiba/Sender experiments/Senders(XL10Gold) T9002(JW1908)#Reaction temparature:37°C|Senders(XL10Gold),T9002(JW1908)@37°C, Sender(&mu;L):Receiver(&mu;L)=500：500,100:1000,10:1000]]====
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====[[Team:Chiba/Sender experiments/Senders(JW1908) T9002(JW1908)#Reaction temparature:37&deg;C|Senders(JW1908), T9002(JW1908)@37&deg;C, Sender(&mu;L):Receiver(&mu;L)=500：500, 100:1000, 10:1000]]====
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====[[Team:Chiba/Sender experiments/Senders(XL10Gold) T9002(JW1908)#Reaction temparature:25°C|Senders(XL10Gold), T9002(JW1908)@25°C, Sender(&mu;L):Receiver(&mu;L)=500：500, 100:1000, 10:1000]]====
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====[[Team:Chiba/Sender experiments/Senders(JW1908) T9002(JW1908)#Reaction temparature:30&deg;C|Senders(JW1908),T9002(JW1908)@30&deg;C,Sender(&mu;L):Receiver(&mu;L)=500：500,100:1000,10:1000]]====
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====[[Team:Chiba/Sender experiments/Senders(XL10Gold) T9002(JW1908)#Reaction temparature:37°C|Senders(XL10Gold),T9002(JW1908)@37&deg;C, Sender(&mu;L):Receiver(&mu;L)=500：500,100:1000,10:1000]]====
 +
 
 +
====[[Team:Chiba/Sender experiments/Senders(XL10Gold) T9002(JW1908)#Reaction temparature:25&deg;C|Senders(XL10Gold), T9002(JW1908)@25&deg;C, Sender(&mu;L):Receiver(&mu;L)=500：500, 100:1000, 10:1000]]====
=== Demo ~Senders~ ===
=== Demo ~Senders~ ===
We experimentally tested the sender genes LuxI and LasI which produce the greatest time difference.
We experimentally tested the sender genes LuxI and LasI which produce the greatest time difference.
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We mixed bacteria (XL10G) transformed with either the LuxI or LasI genes and bacteria (of the BW strain) transformed with the LuxR gene at a 1:1 ratio and visually observed GFP fluorescence.
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We mixed bacteria (XL10Gold) transformed with either the LuxI or LasI genes and bacteria (of the strain JW1908) transformed with the LuxR gene at a 1:1 ratio and visually observed GFP fluorescence.
=== Results ===
=== Results ===
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[[Image:Team-Chiba-demo-mihon.gif|200px|frame|left|'''Fig. 4　Demo''']]<br> Green region: sender=LuxI (50 uL), Red circular region: sender=Las I (50 uL). <br>Receiver=LuxR (50 uL)
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[[Image:Team-Chiba-demo-mihon.gif|200px|frame|left|'''Fig. 4　Demo''']]<br> Green region: sender=LuxI (50 uL), Red circular region:sender culture containing [http://partsregistry.org/Part:BBa_K084007 LasI gene](50 &mu;L).Receiver culture containing [http://partsregistry.org/Part:BBa_T9002 LuxR-plux-gfp gene] (50 &mu;L)
<gallery>
<gallery>
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Image:Team-Chiba-demo-1.JPG|0 h
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Image:Team-Chiba-demo-1.JPG|0 hours
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Image:Team-Chiba-demo-2.JPG|4 h <br>(Lux I GFP detected)
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Image:Team-Chiba-demo-2.JPG|4 hours <br>(LuxI GFP detected)
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Image:Team-Chiba-demo-3.JPG|8 h <br>(Lux I GFP and Las I GFP detected)
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Image:Team-Chiba-demo-3.JPG|8 hours <br>(LuxI GFP and LasI GFP detected)
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</gallery>　
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</gallery>Fig. 5
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-->more about [[Team:Chiba/Demo_experiments:Senders|Demo experiments detail]]
-->more about [[Team:Chiba/Demo_experiments:Senders|Demo experiments detail]]
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#[http://partsregistry.org/Part:BBa_F2620:Specificity BBa_F2620:Specificity]
#[http://partsregistry.org/Part:BBa_F2620:Specificity BBa_F2620:Specificity]
#[http://mic.sgmjournals.org/cgi/content/full/153/12/3923 Paul Williams.:Quorum sensing, communication and cross-kingdom signalling in the bacterial world.Microbiology 153 (2007), 3923-3938]
#[http://mic.sgmjournals.org/cgi/content/full/153/12/3923 Paul Williams.:Quorum sensing, communication and cross-kingdom signalling in the bacterial world.Microbiology 153 (2007), 3923-3938]
 +
#[http://www.pnas.org/content/100/suppl.2/14549.full Michiko E. Taga. Bonnie　L.Bassler.:Chemical communication among bacteria.PNAS.November 25, 2003,'''100'''.suppl.2]

Latest revision as of 11:00, 30 October 2008

Chiba-U.gif
Home The Team The Project Parts Submitted to the Registry Reference Notebook Acknowledgements

Sender Crosstalk

Design

Fig. 1　Sender switch
Fig. 2　AHL varieties

Each species has their own LuxI-type proteins,which synthesize their specific autoinducers, AHLs. AHLs produced by different LuxI-type proteins differ only in the length of the acyl-chain moiety and substitution at position C-3(4).LuxR,which is original for Vibrio fischeri, is activated by its cognate autoinducer, 3OC6HSL. However, LuxR is also activated by non-endogenous molecules, C4HSL, C6HSL, and 3OC12HSL. Activation by non-endogenous molecules requires a higher signal concentration (1),(2). This results in slower activation of receivers, when AHL concentration is increasing.




Experiments

The purpose of this experiment was to create delays in communication time using cross-talk between non-specific signals. We used the following genes for this experiment.

senders (cell: XL10-Gold or JW1908) receiver (cell: JW1908)
*[http://partsregistry.org/Part:BBa_K084007 plac+rbs+LasI] (pSB1AK3)

Tet-lasi chiba.gif

*[http://partsregistry.org/Part:BBa_T9002 BBa_T9002 (Express GFP in response to AHL)] (pSB1A3)

High-Copy-Receiver Chiba.gif

*[http://partsregistry.org/Part:BBa_K084008 plac+rbs+RhlI] (pSB1AK3)

Tet-rhli chiba.gif

*[http://partsregistry.org/Part:BBa_K084009 BBa_K084009]

Tet-rhli chiba.gif

*[http://partsregistry.org/Part:BBa_K084012 plac+rbs+LuxI] (pSB1AK3)

Tet-luxi-cm chiba.gif

*[http://partsregistry.org/Part:BBa_K084014 BBa_K084014]

Tet-luxi chiba.gif

*[http://partsregistry.org/Part:BBa_S03623 BBa_S03623(ptet+rbs+LuxI(LVA))]

Chiba BBa S03623.gif

Details

Method

To characterize quorum sensing crosstalk, constitutive AHL senders were mixed with constitutive receivers and measure fluorescence intensity.

  1. Transformed Senders into E. coli strains (XL10Gold or JW1908) and Receiver into E. coli strain (JW1908).
  2. Inoculated them independently in liquid media. Incubated at 37°C for 12hours.
  3. Mixed them.
  4. Incubated at 37°C or 30°C.
  5. Measured intensity of green fluorescence (485 nm (excitation) and 527 nm (emission)) at regular time intervals.

more details...

Results & Discussion

Fig. 3　senders crosstalk test.senders strain XL10Gold,Receiver strain JW1908.Reaction temparature was 30°C.All measurements are averages from three replicate cultures with error bars representing standard deviations.Labeling:LuxI, RhlI, and LasI means fluorescence induced by AHLs synthesized by [http://partsregistry.org/Part:BBa_K084012 BBa_K084012], [http://partsregistry.org/Part:BBa_K084008 BBa_K084008], and [http://partsregistry.org/Part:BBa_K084007 BBa_K084007] respectively.

Senders(XL10Gold), T9002(JW1908)@30°C, Sender(μL):Receiver(μL)=500：500, 100:1000, 10:1000

When incubated at 30°C,E.coli strain(XL10Gold) transformed with the LasI genes produced more AHL than at 37°C.

(Green fluorescence intensity after incubating for 8 hours was:

163 at 37°C.
245 at 30°C.

3OC12HSL produced by LasI protein(expressed by[http://partsregistry.org/Part:BBa_K084007 BBa_K084007] and 3OC6HSL produced by LuxI protein(expressed by[http://partsregistry.org/Part:BBa_K084012 BBa_K0840012]) both activated LuxR protein and cousese gfp expression.The increase of green fluorescence intensity caused by 3OC12HSL was more gradualy than that caused by 3OC6HSL,time before fluorescence intensity reached at 200 was 2.5 hours longer than that of the 3OC6HSL.

Future plans

  • Reduce quantity of expression of an enzyme composing AHL
  1. Replace medium RBS with weak one.
  2. Alter the copy number of plasmids in the sender into low.
  • To make the slope of transfer curve of our device to be steep:
  1. Introduce Positive Feedback Loop into our genetic circuit.

Other Experiments

Visual Judgement

fluorescence intensity was from 150 to 200 when the 2 mL of reaction culture in a test tube.

Senders(JW1908), T9002(JW1908)@37°C, Sender(μL):Receiver(μL)=500：500, 100:1000, 10:1000

Senders(JW1908),T9002(JW1908)@30°C,Sender(μL):Receiver(μL)=500：500,100:1000,10:1000

Senders(XL10Gold),T9002(JW1908)@37°C, Sender(μL):Receiver(μL)=500：500,100:1000,10:1000

Senders(XL10Gold), T9002(JW1908)@25°C, Sender(μL):Receiver(μL)=500：500, 100:1000, 10:1000

Demo ~Senders~

We experimentally tested the sender genes LuxI and LasI which produce the greatest time difference. We mixed bacteria (XL10Gold) transformed with either the LuxI or LasI genes and bacteria (of the strain JW1908) transformed with the LuxR gene at a 1:1 ratio and visually observed GFP fluorescence.

Results

Fig. 4　Demo

Green region: sender=LuxI (50 uL), Red circular region:sender culture containing [http://partsregistry.org/Part:BBa_K084007 LasI gene](50 μL).Receiver culture containing [http://partsregistry.org/Part:BBa_T9002 LuxR-plux-gfp gene] (50 μL)

0 hours

4 hours
(LuxI GFP detected)

8 hours
(LuxI GFP and LasI GFP detected)

Fig. 5

-->more about Demo experiments detail

references

  1. [http://www3.interscience.wiley.com/journal/119124142/abstract M.K Winson et al.:Construction and analysis of luxCDABE-based plasmid sensors　for investigating N-acyl homoserine lactone-mediated quorum sensing.FEMS Microbiology Letters 163 (1998) 185-192]
  2. [http://partsregistry.org/Part:BBa_F2620:Specificity BBa_F2620:Specificity]
  3. [http://mic.sgmjournals.org/cgi/content/full/153/12/3923 Paul Williams.:Quorum sensing, communication and cross-kingdom signalling in the bacterial world.Microbiology 153 (2007), 3923-3938]
  4. [http://www.pnas.org/content/100/suppl.2/14549.full Michiko E. Taga. Bonnie　L.Bassler.:Chemical communication among bacteria.PNAS.November 25, 2003,100.suppl.2]
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