Team:Chiba/Project/Experiments:Receiver Crosstalk

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__NOTOC__
__NOTOC__
==Receiver Cross-talk==
==Receiver Cross-talk==
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[[Image:Chiba project design Receiver.jpg|left]]
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===Design===
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English:
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-
日本語:AHLを合成するSenderだけではなく、AHLを受け取る側のReceiverを変えれば、その応答時間を変えることができる。そこで私たちは、以下のいくつかの方法を考えた。
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{| style="border:0px;" cellpadding="5px"
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#一種類のSender（AHL<--LuxI）に対して、由来生物の異なるレシーバタンパク質でそれを受信する.
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| width="50%" valign="top" |
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#レシーバータンパク質であるLuxRに変異を入れることで、AHLに対する応答感度を上下させること.
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[[Image:Receiver switch chiba.jpg|frame|left|'''Fig. 1　Crosstalk''']]<br clear=all>
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#レシーバーのコピーナンバーを変える.
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| width="50%" valign="top" |
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[[Image:Table R chiba.gif|frame|left|'''Table 1　LuxR family gene. '''Data modified from M.RIVAS et.al. Biol Res 38: 283-297, (2005)''']]
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[[Image:Lux box Chiba.jpg|frame|left|'''Table 2　Comparison of the predicted Lux box. '''Data modified from A.Fekete et.al. Anal Bioanal Chem 387:455–467,(2007)''']]
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<br clear=all>
 
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{| style="border:0px;" cellpadding="5px"
 
| width="50%" valign="top" |
| width="50%" valign="top" |
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====[[Team:Chiba/AHL Receiver Phase#crosstalk|Quorum-Sensing Crosstalk]]====
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|}
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[[Image:Receiver switch chiba.jpg|frame|left|'''Fig.　''' Crosstalk]]<br clear=all>
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クオラムセンシングにおける、レシーバータンパクを変えてクロストークを起こさせる。<br>
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In this plan, we induce cross-talk of quorum sensing by changing receiver protein. It is known that receiver proteins can work with the stimuli of signaling molecule even from another species of bacteria (See Table. 1).[[Team:Chiba/Project#references|<sup>(1)</sup>]][[Team:Chiba/Project#references|<sup>(2)</sup>]]。<br>
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センダーを変えたときと同様に、他種生物由来のレシーバーでもAHLに応答することは知られている[[Team:Chiba/Project#references|<sup>(1)</sup>]][[Team:Chiba/Project#references|<sup>(2)</sup>]]。<br>
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These endo-genieous combination of signaling molecules and receiver proteins causes the delay of response time and as a result the gene expression triggered by quorum sensing device is to be delayed.
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本来の組み合わせとは異なるAHLを受け取るレシーバーの応答時間は遅くなり、遺伝子発現が遅くなる。
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<br>
<br>
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[[Team:Chiba/AHL Receiver Phase#Quorum Sensing Crosstalk|more about Receiver phase crosstalk]]
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For instance, receiver proteins, RhlR and LasR (from <i>Pseudomonas aeruginosa</i>) is stimulized by 3OC6HSL.
 +
<br clear=all>
 +
 
 +
 
 +
In addition, LuxR protein family activates the expression of gene which is located under a unique sequences of so-called Lux box promotor.(Table. 2)<br>
 +
Strictly speaking, the sequences of Lux box are dependent of the species of bacteria, however, they are quie similar so that we aim the gene expression of endo-geneous LuxR protein family with AHL.
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[[Image:R-family-Crosstalk-Lux Chiba.gif|thumb|left|'''Fig.'''Time Delay Test LuxI-LuxR]]
 
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[[Image:R-family-Crosstalk-Las Chiba.gif|thumb|right|'''Fig.'''Time Delay Test LuxI-LasR]]
 
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[[Image:R-family-Crosstalk-Rhl Chiba.gif|thumb|left|'''Fig.'''Time Delay Test LuxI-RhlR]]
 
<br clear=all>
<br clear=all>
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[[Team:Chiba/AHL Receiver Phase#Quorum Sensing Crosstalk|more about experimental result]]
 
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| width="50%" valign="top" |
 
 +
===Experiment===
 +
This experiment is for induction of cross-talk by changing LuxR protein family.<br>
 +
As AHL sender, the plasmid of Lux I protein from <i>Vibrio fischeri</i> was introduced and 3OC6HSL
 +
is synthesized by the sender.<br>
 +
As AHL Receiver, Receiver plasmid of LuxR protein family and the reporter plasmid of Lux promoter GFP were introduced.
 +
The AHL is produced by the AHL sender and stimulate the AHL receiver, triggering the GFP expression.
 +
We measured the fluorescence intensity of GFP using plate reader for investigation of activity of this cross-talk combination.
 +
The gene circuit used in this section is as follows.
 +
 +
{| class="tbl" |
 +
|-
 +
|
 +
*'''Sender'''
 +
|
 +
*'''Transcriptional regulator'''
 +
|
 +
*'''Reporter'''
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|-
 +
| rowspan="4" |
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*AHL sender[http://partsregistry.org/Part:BBa_S03623 (BBa_S03623) ]
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[[Image:LuxI-sender Chiba.gif|left|]]<br clear=all>
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|
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:*LuxR
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[[Image:LuxR-p15A Chiba.gif]]
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| rowspan="4" |
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:*Plux-GFP[http://partsregistry.org/Part:BBa_J37032 (BBa_J37032)]
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[[Image:Plux-GFP-pMB1 Chiba.gif]]
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|-
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|
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:*RhlR[http://partsregistry.org/Part:BBa_K084004 (BBa_K084004)]
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[[Image:RhlR-p15A Chiba.gif]]
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|-
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|
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:*LasR[http://partsregistry.org/Part:BBa_K084005 (BBa_K084005)]
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[[Image:LasR-p15A Chiba.gif]]
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|-
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|
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:*CinR[http://partsregistry.org/Part:BBa_K084006 (BBa_K084006)]
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[[Image:CinR-p15A Chiba.gif]]
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|}
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===Method===
 +
#Transformed Sender into ''E. coli'' strains (JW1908) and Receivers into ''E. coli'' strain (JW1908).
 +
#Inoculated them independently in liquid media. Incubated at 37c&deg; 12h.
 +
#Inoculated again at 37c&deg; upto about OD600=2.0
 +
#Washed them.
 +
#Mixed them (Sender:Receiver=1000&mu;l:1000&mu;l).
 +
#Incubated at 30c&deg;.
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#Measured intensity of green fluorescence at regular time intervals.
<br clear=all>
<br clear=all>
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=== Demo ~Receivers~ ===
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===Result & Discussion===
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English:
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[[Image:R-family-Crosstalk-Lux Chiba.gif|thumb|left|'''Fig. 2　Time Delay Test Sender:LuxI,Receiver:LuxR''']]
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<BR>日本語:
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[[Image:R-family-Crosstalk-Las Chiba.gif|thumb|left|'''Fig. 3　Time Delay Test Sender:LuxI, Receiver:LasR''']]
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<BR>
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[[Image:R-family-Crosstalk-Rhl Chiba.gif|thumb|left|'''Fig. 4　Time Delay Test Sender:LuxI, Receiver:RhlR''']]
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固体培地中にセンダー[http://partsregistry.org/Part:BBa_S03623 BBa_S03623],(Ptet-LuxI) を混ぜ、固体培地表面にレシーバーのコロニーをN.Cフィルターで移す。
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センダーの作るAHLは培地中を移動し、表面のレシーバーがAHLを一定濃度感知すればGFPを発現
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する。一種のセンダーに対し、様々な種類のレシーバーを用いることで時間差が生じることを確認する。
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用いるレシーバーは・・・
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<br clear=all>
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The diagrams are the time course change of fluorescence intensity of GFP expression indued by various combination of cross-talk using Lux protein family.<br>
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・シグナル自体を分解するAiia を利用する
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LuxR: high GFP expression level (Fig. 2)<br>
 +
LasR: quite low GFP expression level (Fig. 3, 4)<br>
 +
<br>
 +
As summary, in the cross-talk quorum sensing, the expression level is likely to decrease and then we could not recognize the delayed swith of GFP expression in these crosstalk combinatiom.
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・レシーバーの遺伝子回路を含むプラスミドのコピーナンバーの変化
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In this experiment, except LuxR, other R protein family proteins were tagged with LVA tag.
 +
Since LuxI->LasR->PLas and LuxI->RhlR->PRhl curcuit provides low expression level [http://www3.interscience.wiley.com/journal/119124142/abstract (1)], this experimental result is from the LVA tag.
 +
The experiment usig combination of Sender: LuxI (no LAV tag), Receiver: LuxR, LasR, and RhlR (no LAV tag) is furhter necessary to discuss on the current result.
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・レシーバータンパク質であるLuxRに変異を入れる
 
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<BR>
 
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・確認の仕方
 
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<BR>
 
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N.Cフィルターをはった個体培地を37&deg;Cで培養し、時間(30min?)ごとにUVをあててGFPが見えるかチェックする。
 
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<BR>
 
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香取
 
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====Results====
 
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--->more about [[Team:Chiba/Demo_experiments|Demo experiments detail]]
 
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--->more about [[Team:Chiba/Demo_experiments:Receivers|Demo experiments detail]]
 
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<br clear=all>
 
<br clear=all>
<br clear=all>
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===Reference===
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#[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]
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#[http://partsregistry.org/Part:BBa_F2620:Specificity BBa_F2620:Specificity]
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 +
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'''>[[Team:Chiba/Project#Receiver|Back to the project page]]'''
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{| style="color:white;background-color:Maroon" cellpadding="3" cellspacing="3" border="1" bordercolor="white" width="100%" align="center"
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!align="center"|[[Team:Chiba|Home]]
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!align="center"|[[Team:Chiba/Team|The Team]]
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!align="center"|[[Team:Chiba/Project|The Project]]
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!align="center"|[[Team:Chiba/Parts|Parts Submitted to the Registry]]
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!align="center"|[[Team:Chiba/Reference|Reference]]
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!align="center"|[[Team:Chiba/Notebook|Notebook]]
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!align="center"|[[Team:Chiba/Acknowledgements|Acknowledgements]]
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|}

Latest revision as of 06:33, 30 October 2008

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

Receiver Cross-talk

Design

Fig. 1　Crosstalk
Table 1　LuxR family gene. Data modified from M.RIVAS et.al. Biol Res 38: 283-297, (2005)
Table 2　Comparison of the predicted Lux box. Data modified from A.Fekete et.al. Anal Bioanal Chem 387:455–467,(2007)

In this plan, we induce cross-talk of quorum sensing by changing receiver protein. It is known that receiver proteins can work with the stimuli of signaling molecule even from another species of bacteria (See Table. 1).(1)(2)。
These endo-genieous combination of signaling molecules and receiver proteins causes the delay of response time and as a result the gene expression triggered by quorum sensing device is to be delayed.
For instance, receiver proteins, RhlR and LasR (from Pseudomonas aeruginosa) is stimulized by 3OC6HSL.


In addition, LuxR protein family activates the expression of gene which is located under a unique sequences of so-called Lux box promotor.(Table. 2)
Strictly speaking, the sequences of Lux box are dependent of the species of bacteria, however, they are quie similar so that we aim the gene expression of endo-geneous LuxR protein family with AHL.


Experiment

This experiment is for induction of cross-talk by changing LuxR protein family.
As AHL sender, the plasmid of Lux I protein from Vibrio fischeri was introduced and 3OC6HSL is synthesized by the sender.
As AHL Receiver, Receiver plasmid of LuxR protein family and the reporter plasmid of Lux promoter GFP were introduced. The AHL is produced by the AHL sender and stimulate the AHL receiver, triggering the GFP expression. We measured the fluorescence intensity of GFP using plate reader for investigation of activity of this cross-talk combination. The gene circuit used in this section is as follows.

  • Sender
  • Transcriptional regulator
  • Reporter
  • AHL sender[http://partsregistry.org/Part:BBa_S03623 (BBa_S03623) ]
LuxI-sender Chiba.gif
  • LuxR

LuxR-p15A Chiba.gif

  • Plux-GFP[http://partsregistry.org/Part:BBa_J37032 (BBa_J37032)]

Plux-GFP-pMB1 Chiba.gif

  • RhlR[http://partsregistry.org/Part:BBa_K084004 (BBa_K084004)]

RhlR-p15A Chiba.gif

  • LasR[http://partsregistry.org/Part:BBa_K084005 (BBa_K084005)]

LasR-p15A Chiba.gif

  • CinR[http://partsregistry.org/Part:BBa_K084006 (BBa_K084006)]

CinR-p15A Chiba.gif

Method

  1. Transformed Sender into E. coli strains (JW1908) and Receivers into E. coli strain (JW1908).
  2. Inoculated them independently in liquid media. Incubated at 37c° 12h.
  3. Inoculated again at 37c° upto about OD600=2.0
  4. Washed them.
  5. Mixed them (Sender:Receiver=1000μl:1000μl).
  6. Incubated at 30c°.
  7. Measured intensity of green fluorescence at regular time intervals.


Result & Discussion

Fig. 2　Time Delay Test Sender:LuxI,Receiver:LuxR
Fig. 3　Time Delay Test Sender:LuxI, Receiver:LasR
Fig. 4　Time Delay Test Sender:LuxI, Receiver:RhlR


The diagrams are the time course change of fluorescence intensity of GFP expression indued by various combination of cross-talk using Lux protein family.

LuxR: high GFP expression level (Fig. 2)
LasR: quite low GFP expression level (Fig. 3, 4)

As summary, in the cross-talk quorum sensing, the expression level is likely to decrease and then we could not recognize the delayed swith of GFP expression in these crosstalk combinatiom.

In this experiment, except LuxR, other R protein family proteins were tagged with LVA tag. Since LuxI->LasR->PLas and LuxI->RhlR->PRhl curcuit provides low expression level [http://www3.interscience.wiley.com/journal/119124142/abstract (1)], this experimental result is from the LVA tag. The experiment usig combination of Sender: LuxI (no LAV tag), Receiver: LuxR, LasR, and RhlR (no LAV tag) is furhter necessary to discuss on the current result.





Reference

  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]


>Back to the project page

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