Team:Chiba/Project/Experiments:Sender Crosstalk

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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)	*[http://partsregistry.org/Part:BBa_T9002 BBa_T9002 (Express GFP in response to AHL)] (pSB1A3)
*[http://partsregistry.org/Part:BBa_K084008 plac+rbs+RhlI] (pSB1AK3)
*[http://partsregistry.org/Part:BBa_K084009 BBa_K084009]
*[http://partsregistry.org/Part:BBa_K084012 plac+rbs+LuxI] (pSB1AK3)
*[http://partsregistry.org/Part:BBa_K084014 BBa_K084014]
*[http://partsregistry.org/Part:BBa_S03623 BBa_S03623(ptet+rbs+LuxI(LVA))]

Details

Method

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

Transformed Senders into E. coli strains (XL10Gold or JW1908) and Receiver into E. coli strain (JW1908).
Inoculated them independently in liquid media. Incubated at 37°C for 12hours.
Mixed them.
Incubated at 37°C or 30°C.
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

Replace medium RBS with weak one.
Alter the copy number of plasmids in the sender into low.

To make the slope of transfer curve of our device to be steep:

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

[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]
[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]

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 __NOTOC__
 ==Sender Crosstalk==
 === Design===
-[[Image:Sender switch Chiba.jpg|left|thumb]]
+[[Image:Sender switch Chiba.jpg|left|frame|'''Fig. 1　Sender switch''']]
+[[Image:AHL variety chiba.gif|frame|left|'''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[[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.
-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.
-異なる生物はそれぞれに異なるLuxIタイプのタンパク質を持ち、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を合成する。それぞれの生物種のLuxIタイプのタンパク質、それが合成する分子名は以下の表のようである。 (Fig.4).AHLを受け取り応答するLuxRタンパク質はVibrio fischeri由来であり、3OC6HSLに応答する。しかし、他種生物由来のAHLにも応答することが知られており、このとき、より高い濃度のAHLが必要となる(1),(2).AHLがゆっくり溜まっていく時、LuxRは3OC6HSLに対して最も早く応答し、他のAHLに対してはそれよりも遅く応答する。 (冨永)
-[[Image:AHL variety chiba.gif|frame|right|'''Fig.4''' AHL varieties]]
 <br clear="all">
 ===Experiments===
-センダーの違いによるディレイをみることをめざし，次の表に示すセンダーとレシーバを別々の培地で培養@37℃,12h後（stationaryに達したら？対数増殖機の細胞の分裂によるsender量増加を防ぐため？），それぞれを混ぜ，３０℃で培養し，一定時間ごとの蛍光強度（__nm）を測定した。
+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.
 {| class="tbl" |
 |-
-|'''senders''' (cell: XL10-Gold)
+|'''senders''' (cell: XL10-Gold or JW1908)
 |'''receiver''' (cell: JW1908)
 |-
-| *[http://partsregistry.org/Part:BBa_K084007 plac+rbs+LasI] (plasmid?)
+| *[http://partsregistry.org/Part:BBa_K084007 plac+rbs+LasI] (pSB1AK3)
 [[Image:Tet-lasi chiba.gif]]
-| rowspan="3" | *[http://partsregistry.org/Part:BBa_T9002 BBa_T9002 (Express GFP in response to AHL)] (plasmid?)
+| rowspan="6" | *[http://partsregistry.org/Part:BBa_T9002 BBa_T9002 (Express GFP in response to AHL)] (pSB1A3)
-[[Image:Reporter 9002 chiba.gif]]
+[[Image:High-Copy-Receiver Chiba.gif]]
 |-
-| *[http://partsregistry.org/Part:BBa_K084008 plac+rbs+RhlI] (plasmid?)
+| *[http://partsregistry.org/Part:BBa_K084008 plac+rbs+RhlI] (pSB1AK3)
 [[Image:Tet-rhli chiba.gif]]
 |-
-| *[http://partsregistry.org/Part:BBa_K084012 plac+rbs+LuxI] (plasmid?)
+| *[http://partsregistry.org/Part:BBa_K084009 BBa_K084009]
+[[Image:Tet-rhli chiba.gif]]
+|-
+| *[http://partsregistry.org/Part:BBa_K084012 plac+rbs+LuxI] (pSB1AK3)
+[[Image:Tet-luxi-cm chiba.gif]]
+|-
+| *[http://partsregistry.org/Part:BBa_K084014 BBa_K084014]
 [[Image:Tet-luxi chiba.gif]]
+|-
+| *[http://partsregistry.org/Part:BBa_S03623 BBa_S03623(ptet+rbs+LuxI(LVA))]
+[[Image:Chiba_BBa_S03623.gif‎]]
 |}
+===Details===
+====Method====
+To characterize quorum sensing crosstalk, constitutive AHL senders were mixed with constitutive receivers and measure fluorescence intensity.
+#Transformed Senders into ''E. coli'' strains (XL10Gold or JW1908) and Receiver into ''E. coli'' strain (JW1908).
+#Inoculated them independently in liquid media. Incubated at 37&deg;C for 12hours.
+#Mixed them.
+#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.
+[[Team:Chiba/protocol/phenotype/timedelay|more details...]]
-===Results&Discussion===
+===Results & Discussion===
 [[image:senders_crosstalk_chiba_01.gif|frame|
-'''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.]]
+'''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.]]
-====[[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]]====
+====[[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]]====
-*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）
+When incubated at 30&deg;C,E.coli strain(XL10Gold) transformed with the LasI genes produced more AHL than at 37&deg;C.
-*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。
+(Green fluorescence intensity after incubating for 8 hours was:
-: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。
+::163 at 37&deg;C.
-OC6HSL,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）。
+::245 at 30&deg;C.
-(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。) <??
+OC12HSL 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
+#Replace medium RBS with weak one.
+#Alter the copy number of plasmids in the sender into low.
+*To make the slope of transfer curve of our device to be steep:
+#Introduce Positive Feedback Loop into our genetic circuit.
-===Details===
+===Other Experiments===
-====Method====
+====Visual Judgement====
-.Crosstalk test
-To characterize quorum sensing crosstalk, constitutive AHL senders were mixed with constitutive receivers and measure fluorescence intensity.
+fluorescence intensity was from 150 to 200 when the 2 mL of reaction culture in a test tube.
-#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
-#Mixed them.
-#Incubated at 30°C.
-#Measured intensity of green fluorescence at regular time intervals.
-[[Team:Chiba/protocol/phenotype/timedelay|more details...]]
+====[[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]]====
+====[[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]]====
-===Other Experiments===
+====[[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]]====
-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。
+====[[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]]====
---[[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]]====
+=== Demo ~Senders~ ===
-*CinI+LVAとLuxRのクロストークはどの菌数比でもおこらない。
+We experimentally tested the sender genes LuxI and LasI which produce the greatest time difference.
-*RhlIが（RhlI+LVA、LasI、CinI+LVAの中で）一番クロストークする。
+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.
-*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]]====
+=== Results ===
-*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]]====
+[[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)
-*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）
+<gallery>
+Image:Team-Chiba-demo-1.JPG|0 hours
+Image:Team-Chiba-demo-2.JPG|4 hours <br>(LuxI GFP detected)
+Image:Team-Chiba-demo-3.JPG|8 hours <br>(LuxI GFP and LasI GFP detected)
+</gallery>Fig. 5
+-->more about [[Team:Chiba/Demo_experiments:Senders|Demo experiments detail]]
-====[[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]]====
+==references==
-*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。
+#[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]
-（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。）
+#[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://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]