Team:Chiba/Sender experiments

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!align="center"|[[Team:Chiba/Project|The Project]]
!align="center"|[[Team:Chiba/Project|The Project]]
!align="center"|[[Team:Chiba/Parts|Parts Submitted to the Registry]]
!align="center"|[[Team:Chiba/Parts|Parts Submitted to the Registry]]
 +
!align="center"|[[Team:Chiba/Reference|Reference]]
!align="center"|[[Team:Chiba/Notebook|Notebook]]
!align="center"|[[Team:Chiba/Notebook|Notebook]]
 +
!align="center"|[[Team:Chiba/Acknowledgements|Acknowledgements]]
|}
|}
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==Quorum-Sensing Cross-talk==
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__NOTOC__
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====Purpose====
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==Signal molecule sender==
 +
 
 +
==Design==
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 +
[[Image:Sender switch Chiba.jpg|left|]]
 +
 
 +
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.
 +
 
 +
<br clear=all>
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 +
[[Image:AHL variety chiba.gif|frame|'''Fig.4''' AHL varieties]]
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 +
 
 +
==Experiment==
To Confirm that communication using non-endogenous molecules results in slower
To Confirm that communication using non-endogenous molecules results in slower
activation of receivers.
activation of receivers.
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===Method===
===Method===
 +
1.Crosstalk test
 +
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(JW1908/XL10GOLD) and Receiver into E.coli strain(JW1908).
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#Transformed Senders into E.coli strains(XL10GOLD) and Receiver into E.coli strain(JW1908).
#Inoculated them independently in liquid media. Incubated at 37°C 12h
#Inoculated them independently in liquid media. Incubated at 37°C 12h
#Mixed them.
#Mixed them.
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#Measured intensity of green fluorescence at regular time intervals.
#Measured intensity of green fluorescence at regular time intervals.
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===Result===
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[[Team:Chiba/protocol/phenotype/timedelay|more details...]]
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====1.Crosstalk test at 30°C ====
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[[image:senders_crosstalk_chiba_01.gif|frame|left|
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-
'''Fig.''' senders crosstalk test.senders strain XL10Gold,Receiver strain JW1908.Reaction temparature was 30°C.Labeling:LuxI,RhlI,LasI means fluorescence induced by AHLs synthesized by LuxI,RhlI,LasI respectively.]]<br clear="all">
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-
*RhlIとLuxIでは、GFP inductionにかかる時間はほとんど同じであった。
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===Result and Discussion===
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*LasIは、GFP inductionが他より約2時間遅れた。
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目視実験
 +
*試験管内の培養液(2mL)が、緑色を呈しているときの蛍光強度：150～200。
 +
--[[User:Masahiro|Masahiro]] 07:16, 29 October 2008 (UTC)
 +
 
 +
====[[Team:Chiba/Sender experiments/Senders(JW1908) T9002(JW1908)#Reaction temparature:37°C|Senders(JW1908),T9002(JW1908)@37°C菌数(μL)、Sender:Receiver＝500：500,100:1000,10:1000]]====
 +
*CinI+LVAとLuxRのクロストークはどの菌数比でもおこらない。
 +
*RhlIが（RhlI+LVA、LasI、CinI+LVAの中で）一番クロストークする。
 +
*Sender同士のクロストークの時差（蛍光強度200に達するまでの時間）は、1時間以下の範囲でしか発生しなかった。
 +
 
 +
====[[Team:Chiba/Sender experiments/Senders(JW1908) T9002(JW1908)#Reaction temparature:30°C|Senders(JW1908),T9002(JW1908)@30°C菌数(μL)、Sender:Receiver＝500：500,100:1000,10:1000]]====
 +
*特に37°Cで行ったときと差はない
 +
*BWはクオラムセンシングをしやすい株なので、Senderを変えてもたいした時差が見られなかった。
 +
*次回からはSender側の株をXL10Gに代えて実験を行う。
 +
 
 +
====[[Team:Chiba/Sender experiments/Senders(XL10Gold) T9002(JW1908)#Reaction temparature:37°C|Senders(XL10Gold),T9002(JW1908)@37°C菌数(μL)、Sender:Receiver＝500：500,100:1000,10:1000]]====
 +
*SenderをBW株にして行った実験よりも、8時間後の蛍光強度が平均で200くらい落ちた。
 +
*この条件ではLasIのクロストークは起こりにくい。
 +
:（８ｈ後の最終蛍光強度（菌比１：１）、LasI：LuxI(Ptet)：RhlI：RhlI+LVA＝163：267：394：325）
 +
 
 +
====[[Team:Chiba/Sender experiments/Senders(XL10Gold) T9002(JW1908)#Reaction temparature:30°C|Senders(XL10Gold),T9002(JW1908)@30°C菌数(μL)、Sender:Receiver＝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℃：30℃＝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。
 +
 
 +
====[[Team:Chiba/Sender experiments/Senders(XL10Gold) T9002(JW1908)#Reaction temparature:25°C|Senders(XL10Gold),T9002(JW1908)@25°C菌数(μL)、Sender:Receiver＝500：500,100:1000,10:1000]]====
 +
*どのSenderもGFP強度20～50の範囲。ネガコン（T9002のみ）の値、40前後と変わらず。
 +
（ただし、静置培養のため、しんとう培養した30℃や37℃とは条件が違う。）
 +
 
 +
--[[User:Yoshimi|Yoshimi]] 06:14, 29 October 2008 (UTC)
 +
 
 +
--[[User:Masahiro|Masahiro]] 07:47, 29 October 2008 (UTC)
 +
 
 +
===Conclusion===
 +
    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。
 +
 
 +
[[image:senders_crosstalk_chiba_01.gif|frame|left|
 +
'''Fig.''' 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.]]<br clear="all">
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====2.Demonstration====
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--[[User:Masahiro|Masahiro]] 09:30, 29 October 2008 (UTC)
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'''>[[Team:Chiba/Project#Sender|Back to the project page]]'''
 
{| style="color:white;background-color:Maroon" cellpadding="3" cellspacing="3" border="1" bordercolor="white" width="100%" align="center"
{| style="color:white;background-color:Maroon" cellpadding="3" cellspacing="3" border="1" bordercolor="white" width="100%" align="center"
!align="center"|[[Team:Chiba|Home]]
!align="center"|[[Team:Chiba|Home]]
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!align="center"|[[Team:Chiba/Project|The Project]]
!align="center"|[[Team:Chiba/Project|The Project]]
!align="center"|[[Team:Chiba/Parts|Parts Submitted to the Registry]]
!align="center"|[[Team:Chiba/Parts|Parts Submitted to the Registry]]
 +
!align="center"|[[Team:Chiba/Reference|Reference]]
!align="center"|[[Team:Chiba/Notebook|Notebook]]
!align="center"|[[Team:Chiba/Notebook|Notebook]]
 +
!align="center"|[[Team:Chiba/Acknowledgements|Acknowledgements]]
|}
|}

Latest revision as of 12:14, 29 October 2008

Chiba-U.gif

Home The Team The Project Parts Submitted to the Registry Reference Notebook Acknowledgements


Signal molecule sender

Design

Sender switch Chiba.jpg

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.


Fig.4 AHL varieties


Experiment

To Confirm that communication using non-endogenous molecules results in slower activation of receivers.

冨永

Fig.5 Senders crosstalk

Senders

  • [http://partsregistry.org/Part:BBa_K084007 plac+rbs+LasI]

Tet-lasi chiba.gif

  • [http://partsregistry.org/Part:BBa_K084008 plac+rbs+RhlI]

Tet-rhli chiba.gif

  • [http://partsregistry.org/Part:BBa_K084012 plac+rbs+LuxI]

Tet-luxi chiba.gif

Receiver

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

Reporter 9002 chiba.gif

Method

1.Crosstalk test

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 12h
  3. Mixed them.
  4. Incubated at 30°C.
  5. Measured intensity of green fluorescence at regular time intervals.

more details...

Result and Discussion

目視実験

  • 試験管内の培養液(2mL)が、緑色を呈しているときの蛍光強度：150～200。

--Masahiro 07:16, 29 October 2008 (UTC)

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

  • CinI+LVAとLuxRのクロストークはどの菌数比でもおこらない。
  • RhlIが（RhlI+LVA、LasI、CinI+LVAの中で）一番クロストークする。
  • Sender同士のクロストークの時差（蛍光強度200に達するまでの時間）は、1時間以下の範囲でしか発生しなかった。

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

  • 特に37°Cで行ったときと差はない
  • BWはクオラムセンシングをしやすい株なので、Senderを変えてもたいした時差が見られなかった。
  • 次回からはSender側の株をXL10Gに代えて実験を行う。

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

  • SenderをBW株にして行った実験よりも、8時間後の蛍光強度が平均で200くらい落ちた。
  • この条件ではLasIのクロストークは起こりにくい。
（８ｈ後の最終蛍光強度（菌比１：１）、LasI：LuxI(Ptet)：RhlI：RhlI+LVA＝163：267：394：325）

Senders(XL10Gold),T9002(JW1908)@30°C菌数(μL)、Sender:Receiver＝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℃：30℃＝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。

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

  • どのSenderもGFP強度20～50の範囲。ネガコン（T9002のみ）の値、40前後と変わらず。

（ただし、静置培養のため、しんとう培養した30℃や37℃とは条件が違う。）

--Yoshimi 06:14, 29 October 2008 (UTC)

--Masahiro 07:47, 29 October 2008 (UTC)

Conclusion

   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。
Fig. 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.

--Masahiro 09:30, 29 October 2008 (UTC)


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