Team:Chiba/Project/Experiments:Receiver Crosstalk

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[[Image:Receiver switch chiba.jpg|frame|left|'''Fig.　''' Crosstalk]]<br clear=all>
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[[Image:Receiver switch chiba.jpg|frame|left|'''Fig. 1　Crosstalk''']]<br clear=all>
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[[Image:Table R chiba.gif|frame|left|'''Table.''' LuxR family gene '''Data modified from M.RIVAS et.al. Biol Res 38: 283-297, (2005)''']]
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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|'''Fig.''' Comparison of the predicted Lux box. '''Data modified from A.Fekete et.al. Anal Bioanal Chem 387:455–467,(2007)''']]
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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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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>
+
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>
-
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>
+
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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異なる生物はそれぞれに異なるLuxRタイプのタンパク質を持ち、それぞれアシル鎖の長さ、あるいはC-3位の置換基が異なる種類のAHLに応答する。生物種によって異なるAHLと、それに応答するLuxRタイプのタンパク質はTableに示すとおりである。
+
For instance, receiver proteins, RhlR and LasR (from <i>Pseudomonas aeruginosa</i>) is stimulized by 3OC6HSL.
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Pseudomonas aeruginosa由来のRhlR,LasRも3OC6HSLを受け取ることがわかっている。
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<br clear=all>
<br clear=all>
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また、LuxR protein familyはLux boxという特定の配列を持ったプロモーター下にある遺伝子の発現を活性化する。(Fig. )
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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>
-
Lux boxも生物種によって配列が異なるが、ほとんど変わらない配列を持っているために違った生物種のLuxR protein familyであっても、AHLが存在下で遺伝子発現を活性化すると考えられる。
+
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.
<br clear=all>
<br clear=all>
===Experiment===
===Experiment===
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LuxR protein familyを変えてクロストークさせる実験を行った。
+
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
-
AHL senderとしてVibrio fischeri由来のLuxIを常に発現させ、3OC6HSLを合成させる。
+
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.
-
AHL ReceiverとしてLuxR protein familyを発現させるReceiver plasmidと,Lux promoter下にGFPを含むレポータープラスミドをダブルトランスフォーメーションさせた。
+
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.
-
AHL senderによって合成されたAHLをAHL receiverが受取り、Lux promoter下のGFPが発現する。
+
The gene circuit used in this section is as follows.
-
 
+
-
蛍光強度を蛍光リーダーを使ってGFPの発現を計測し、遺伝子発現の活性を調べた。
+
-
 
+
-
使った遺伝子回路は以下の通りある。
+
 +
{| class="tbl" |
 +
|-
 +
|
*'''Sender'''
*'''Sender'''
-
**AHL autoinucer[http://partsregistry.org/Part:BBa_S03623 (BBa_S03623) ]
+
|
-
 
+
*'''Transcriptional regulator'''
-
 
+
|
-
 
+
*'''Reporter'''
 +
|-
 +
| rowspan="4" |
 +
*AHL sender[http://partsregistry.org/Part:BBa_S03623 (BBa_S03623) ]
[[Image:LuxI-sender Chiba.gif|left|]]<br clear=all>
[[Image:LuxI-sender Chiba.gif|left|]]<br clear=all>
-
 
+
|
-
*'''Transcriptional regulator'''
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:*LuxR
:*LuxR
[[Image:LuxR-p15A Chiba.gif]]
[[Image:LuxR-p15A Chiba.gif]]
 +
| rowspan="4" |
 +
:*Plux-GFP[http://partsregistry.org/Part:BBa_J37032 (BBa_J37032)]
 +
[[Image:Plux-GFP-pMB1 Chiba.gif]]
 +
|-
 +
|
:*RhlR[http://partsregistry.org/Part:BBa_K084004 (BBa_K084004)]
:*RhlR[http://partsregistry.org/Part:BBa_K084004 (BBa_K084004)]
[[Image:RhlR-p15A Chiba.gif]]
[[Image:RhlR-p15A Chiba.gif]]
 +
|-
 +
|
:*LasR[http://partsregistry.org/Part:BBa_K084005 (BBa_K084005)]
:*LasR[http://partsregistry.org/Part:BBa_K084005 (BBa_K084005)]
[[Image:LasR-p15A Chiba.gif]]
[[Image:LasR-p15A Chiba.gif]]
 +
|-
 +
|
:*CinR[http://partsregistry.org/Part:BBa_K084006 (BBa_K084006)]
:*CinR[http://partsregistry.org/Part:BBa_K084006 (BBa_K084006)]
[[Image:CinR-p15A Chiba.gif]]
[[Image:CinR-p15A Chiba.gif]]
-
 
+
|}
-
*'''Reporter'''
+
-
:*Plux-GFP[http://partsregistry.org/Part:BBa_J37032 (BBa_J37032)]
+
-
[[Image:Plux-GFP-pMB1 Chiba.gif]]
+
-
 
+
===Method===
===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;.
 +
#Measured intensity of green fluorescence at regular time intervals.
 +
<br clear=all>
 +
===Result & Discussion===
 +
[[Image:R-family-Crosstalk-Lux Chiba.gif|thumb|left|'''Fig. 2　Time Delay Test Sender:LuxI,Receiver:LuxR''']]
 +
[[Image:R-family-Crosstalk-Las Chiba.gif|thumb|left|'''Fig. 3　Time Delay Test Sender:LuxI, Receiver:LasR''']]
 +
[[Image:R-family-Crosstalk-Rhl Chiba.gif|thumb|left|'''Fig. 4　Time Delay Test Sender:LuxI, Receiver:RhlR''']]
-
[[Team:Chiba/AHL Receiver Phase#Quorum Sensing Crosstalk|more about Receiver phase crosstalk]]
 
-
 
-
===Result & Discussion===
 
-
[[Image:R-family-Crosstalk-Lux Chiba.gif|thumb|left|'''Fig.'''Time Delay Test LuxI-LuxR]]
 
-
[[Image:R-family-Crosstalk-Las Chiba.gif|thumb|left|'''Fig.'''Time Delay Test LuxI-LasR]]
 
-
[[Image:R-family-Crosstalk-Rhl Chiba.gif|thumb|left|'''Fig.'''Time Delay Test LuxI-RhlR]]
 
<br clear=all>
<br clear=all>
-
[[Team:Chiba/AHL Receiver Phase#Quorum Sensing Crosstalk|more about experimental result]]
 
 +
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>
-
<br clear=all>
+
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.
-
=== Demo ~Receivers~ ===
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In this experiment, except LuxR, other R protein family proteins were tagged with LVA tag.
-
English:
+
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.
-
<BR>日本語:
+
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.
-
<BR>
+
-
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。
+
-
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
+
-
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。
+
-
用いるレシーバーは・・・
 
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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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・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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・レシーバータンパク質であるLuxRに変異を入れる
 
-
　　　　　
 
-
<BR>
 
-
・確認の仕方
 
-
<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。
 
-
<BR>
 
-
香取
 
-
====Results====
 
-
--->more about [[Team:Chiba/Demo_experiments|Demo experiments detail]]
 
-
 
-
--->more about [[Team:Chiba/Demo_experiments:Receivers|Demo experiments detail]]
 
<br clear=all>
<br clear=all>
 +
 +
===Reference===
 +
#[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]
 +
#[http://partsregistry.org/Part:BBa_F2620:Specificity BBa_F2620:Specificity]
 +
'''>[[Team:Chiba/Project#Receiver|Back to the project page]]'''
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!align="center"|[[Team:Chiba|Home]]
!align="center"|[[Team:Chiba|Home]]

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
Retrieved from "http://2008.igem.org/Team:Chiba/Project/Experiments:Receiver_Crosstalk"