Team:Chiba/Project/Experiments:Sender Crosstalk

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(Difference between revisions)
(→Senders(XL10Gold), T9002(JW1908)@30°C, Sender(μL):Receiver(μL)=500：500, 100:1000, 10:1000)
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*AHL合成酵素の発現量を減らし,AHL生産速度を遅くする.
*AHL合成酵素の発現量を減らし,AHL生産速度を遅くする.
#Replace medium RBS with weak one.
#Replace medium RBS with weak one.
-
#コピーナンバーをlowにする.
+
#Alter the copy number of plasmids in the sender into low.
*transfer curveをシグモイダルに近づける.
*transfer curveをシグモイダルに近づける.

Revision as of 04:09, 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.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.




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) 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 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,LasI means fluorescence induced by AHLs synthesized by LuxI,RhlI,LasI respectively.

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

  • 37°Cで行った実験と比べ、LasIが活性。（８ｈ後の蛍光強度、37°C：30°C=163：245）
  • LasとLux(Plac)１：１で蛍光強度200に達するまでの時間に2．5時間の時差がみられた。
  • 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。
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。

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）。 (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。) <??

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:

Future plans

  • AHL合成酵素の発現量を減らし,AHL生産速度を遅くする.
  1. Replace medium RBS with weak one.
  2. Alter the copy number of plasmids in the sender into low.
  • transfer curveをシグモイダルに近づける.
  1. Positive Feedback Loopを導入する.

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

  • Among RhlI, LasI, and CinI+LVA, RhlI+LVA is the easist to cross-talk with LuxR
  • Delay time by cross-talk among senders are generated only a range of one hour to amount

to 200 of fluorescence intensity.

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~

I experimentally tested the sender genes LuxI and LasI which produce the greatest time difference. 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.

Results

Fig.4 Demo

Green region: sender=LuxI (50 uL), Red circular region: sender=Las I (50 uL).
Receiver=LuxR (50 uL)

0 h

4 h
(Lux I GFP detected)

8 h
(Lux I GFP and Las I GFP detected)

　


-->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]
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