Team:Chiba/Demo experiments

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[[Image:Chiba-U.gif]]
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{| style="color:white;" cellpadding="3" cellspacing="3" border="0" width="100%" align="center" class="menu" |
!align="center"|[[Team:Chiba|Home]]
!align="center"|[[Team:Chiba|Home]]
!align="center"|[[Team:Chiba/Team|The Team]]
!align="center"|[[Team:Chiba/Team|The Team]]
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!align="center"|[[Team:Chiba/Acknowledgements|Acknowledgements]]
!align="center"|[[Team:Chiba/Acknowledgements|Acknowledgements]]
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__NOTOC__
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== Demo Experiment ~Senders~ ==
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== Demonstration Experiment ~Senders~ ==
===Method===
===Method===
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#プレ培養
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#Pre-culture
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##以下のグリストを各培養液2mL中にPick。
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##Picked and cultured the following glycerol stocks in 2mL of LB:
-
###LB-Amp, [http://partsregistry.org/Part:BBa_T9002 BBa_T9002], (BW)
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###LB-Amp, [http://partsregistry.org/Part:BBa_T9002 BBa_T9002], (JW1908)
###LB-Amp+0.2%Glu, [http://partsregistry.org/Part:BBa_K084012 BBa_K084012]([http://partsregistry.org/Part:BBa_J04500 plac+rbs]+[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0161 LuxI(no LVA)]), (XL10G)
###LB-Amp+0.2%Glu, [http://partsregistry.org/Part:BBa_K084012 BBa_K084012]([http://partsregistry.org/Part:BBa_J04500 plac+rbs]+[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0161 LuxI(no LVA)]), (XL10G)
###LB-Amp+0.2%Glu, [http://partsregistry.org/Part:BBa_K084007 BBa_K084007]([http://partsregistry.org/Part:BBa_J04500 plac+rbs]+[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0178 LasI(no LVA)]), (XL10G)
###LB-Amp+0.2%Glu, [http://partsregistry.org/Part:BBa_K084007 BBa_K084007]([http://partsregistry.org/Part:BBa_J04500 plac+rbs]+[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0178 LasI(no LVA)]), (XL10G)
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##37&deg;Cでしんとう培養。12h
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##Cultured at 37&deg;C for 12h.
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#本培養
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#Culture
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##プレ培養液を6.25%ずつ各培養液に添加する。
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##Added 6.25% each of the pre-cultures to new LB medium.
###LB-Amp, [http://partsregistry.org/Part:BBa_T9002 BBa_T9002]
###LB-Amp, [http://partsregistry.org/Part:BBa_T9002 BBa_T9002]
###LB-Amp+0.2%Glu, [http://partsregistry.org/Part:BBa_K084012 BBa_K084012]([http://partsregistry.org/Part:BBa_J04500 plac+rbs]+[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0161 LuxI(no LVA)]), [http://partsregistry.org/Part:BBa_K084007 BBa_K084007]([http://partsregistry.org/Part:BBa_J04500 plac+rbs]+[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0178 LasI(no LVA)])
###LB-Amp+0.2%Glu, [http://partsregistry.org/Part:BBa_K084012 BBa_K084012]([http://partsregistry.org/Part:BBa_J04500 plac+rbs]+[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0161 LuxI(no LVA)]), [http://partsregistry.org/Part:BBa_K084007 BBa_K084007]([http://partsregistry.org/Part:BBa_J04500 plac+rbs]+[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0178 LasI(no LVA)])
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##37&deg;Cでしんとう培養。4~5h。
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##Cultured at 37&deg;C for 4~5h。
#Wash
#Wash
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##遠心管50mL中に10mLずつ培養液を移す。
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##Transfer 10mL each of the culture to 50mL centrifuge tubes.
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##20&deg;C,3600rpmで6min遠心後、上澄みを捨てる。
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##Centrifuged for 6min at 3600rpm,20&deg;C and discarded the supernatant.
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##各遠心管にLB-Ampを加える。
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##Added LB-Amp to each centrifuge tube:
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###[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]が入った各遠心管に10mL
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###10mL to the tube that contains [http://partsregistry.org/Part:BBa_T9002 BBa_T9002]
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###[http://partsregistry.org/Part:BBa_K084012 BBa_K084012], [http://partsregistry.org/Part:BBa_K084007 BBa_K084007]が入った各遠心管に5mL
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###5mL to the tube that contains [http://partsregistry.org/Part:BBa_K084012 BBa_K084012], [http://partsregistry.org/Part:BBa_K084007 BBa_K084007]
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##[http://partsregistry.org/Part:BBa_K084012 BBa_K084012], [http://partsregistry.org/Part:BBa_K084007 BBa_K084007]が入った遠心管を20&deg;C,3600rpmで6min遠心。上澄みを捨てる。
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##Centrifuged for 6min, 3600rpm at 20&deg;C the tube containing [http://partsregistry.org/Part:BBa_K084012 BBa_K084012], [http://partsregistry.org/Part:BBa_K084007 BBa_K084007] and discarded the supernatant.
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##[http://partsregistry.org/Part:BBa_K084012 BBa_K084012], [http://partsregistry.org/Part:BBa_K084007 BBa_K084007]が入った各遠心管にLB-Ampを5mL添加
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##10mL to the tube that contains [http://partsregistry.org/Part:BBa_K084012 BBa_K084012], [http://partsregistry.org/Part:BBa_K084007 BBa_K084007]
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##[http://partsregistry.org/Part:BBa_K084012 BBa_K084012], [http://partsregistry.org/Part:BBa_K084007 BBa_K084007]が入った遠心管を20&deg;C,3600rpmで6min遠心。上澄みを捨てる。
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##Centrifuged for 6min, 3600rpm at 20&deg;C the tube containing [http://partsregistry.org/Part:BBa_K084012 BBa_K084012], [http://partsregistry.org/Part:BBa_K084007 BBa_K084007] and discarded the supernatant.
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##[http://partsregistry.org/Part:BBa_K084012 BBa_K084012], [http://partsregistry.org/Part:BBa_K084007 BBa_K084007]が入った各遠心管にLB-Ampを10mLずつ添加
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##5mL to the tube that contains [http://partsregistry.org/Part:BBa_K084012 BBa_K084012], [http://partsregistry.org/Part:BBa_K084007 BBa_K084007]
#Mix
#Mix
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##Senderの[http://partsregistry.org/Part:BBa_K084012 BBa_K084012][http://partsregistry.org/Part:BBa_K084007 BBa_K084007]を、それぞれ1:1で[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]と混ぜる。
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##Mixed the sender cells [http://partsregistry.org/Part:BBa_K084012 BBa_K084012] and [http://partsregistry.org/Part:BBa_K084007 BBa_K084007] both with [http://partsregistry.org/Part:BBa_T9002 BBa_T9002] at a 1:1 ratio.
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##96穴シャロウプレートに100&mu;Lずつ入れていく。入れ方は図の通り。
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##Added 100&mu;L each to a 96-well shallow plate (as shown in the figure).
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###緑の部分に[http://partsregistry.org/Part:BBa_K084012 BBa_K084012]:[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]=1:1
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###Green part is [http://partsregistry.org/Part:BBa_K084012 BBa_K084012]:[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]=1:1
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###赤の部分に[http://partsregistry.org/Part:BBa_K084007 BBa_K084007]:[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]=1:1
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###Red part is [http://partsregistry.org/Part:BBa_K084007 BBa_K084007]:[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]=1:1
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###無地の部分に[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]のみ
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###Uncolored part is [http://partsregistry.org/Part:BBa_T9002 BBa_T9002] alone.
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#培養&観察
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#Culture and observe results
=== Results ===
=== Results ===
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結果は以下の通り。
 
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[[Image:Team-Chiba-demo-mihon.gif|200px]] *緑部分:LuxI *赤部分:LasI
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[[Image:Team-Chiba-demo-mihon.gif|200px]] Green region: sender=LuxI,
 +
Red circular region: sender=Las I.
<gallery>
<gallery>
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Image:Team-Chiba-demo-2.JPG|4 h <br>(Lux I GFP detected)
Image:Team-Chiba-demo-2.JPG|4 h <br>(Lux I GFP detected)
Image:Team-Chiba-demo-3.JPG|8 h <br>(Lux I GFP and Las I GFP detected)
Image:Team-Chiba-demo-3.JPG|8 h <br>(Lux I GFP and Las I GFP detected)
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</gallery> 
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</gallery>
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1枚目の写真は開始点。2枚目はLuxIの部分だけGFPを肉眼(蛍光灯光)で確認。3枚目はLuxIとLasI両方を確認したものです。
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LuxI GFP is detected at 4h following mixing while LasI GFP is detected
 +
after 8h, thus successfully demonstrating time-delay depending on the
 +
sender used.
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(それぞれ0時間、4時間、8時間後の映像。液量が100μLと少ないため、今までの実験(1000μL)での結果よりもGFP発現に時間がかかった。・・・コレ書かない方がいいのかなあ。)
 
--[[User:Yoshimi|Yoshimi]] 13:41, 29 October 2008 (UTC)
--[[User:Yoshimi|Yoshimi]] 13:41, 29 October 2008 (UTC)
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== Demo Experiment ~Receivers~ ==
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== Demonstration Experiment ~Receivers~ ==
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===method-菌数ふり実験===
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===Varying bacterial numbers: method===
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#Receiver(T9002)のプレ培養
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#Receiver(T9002) pre-incubation
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##Receiver:[http://partsregistry.org/Part:BBa_T9002 BBa_T9002](BW)をLB-Amp 2ml培養(37&deg;C,12h)
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##Receiver:[http://partsregistry.org/Part:BBa_T9002 BBa_T9002](JW1908)was cultured in 2mL LB-Amp. (37&deg;C,12h)
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##プレ培養したReceiver([http://partsregistry.org/Part:BBa_T9002 BBa_T9002](BW))をコロニーが1000個くらいになるようにプレートにまく。
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##Pre-incubated Receiver([http://partsregistry.org/Part:BBa_T9002 BBa_T9002](JW1908))was plated so as to produce about 1000 colonies.
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#Sender(S03623)のプレ培養
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#Sender(S03623) pre-incubation
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##Sender:[http://partsregistry.org/Part:BBa_S03623 BBa_S03623](BW)をLB-Amp 50ml遠心管で10ml培養(37&deg;C,12h)(1,2の2本)
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##Sender:[http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908) was cultured in 50mL  entrifuge tubes in 10mL of LB-Amp (37&deg;C,12h)(2tubes)
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#senderのWash
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#Sender Wash
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##培養液([http://partsregistry.org/Part:BBa_T9002 BBa_T9002](BW))が入った遠心管(2本)を20&deg;C,3600rpmで6min遠心後、上澄みを捨てる。
+
##Centrifuged 2 tubes containing([http://partsregistry.org/Part:BBa_T9002 BBa_T9002](JW1908))at 20&deg;C,3600rpm for 6min and discarded supernatant.
-
##各遠心管にLB-Amp10mlを加える。
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##Added 10mL LB-Amp to each tube.
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##同様の操作をあと2回行った。
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##Repeated wash twice.
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#固体プレート作り
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#Creating bacterial plates
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##LB-Ampで12hプレ培養したSender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](BW))の培養液-1(10ml)とLB-Amp-agar(50&deg;C)(10ml)を混ぜてsender入りの固体培地-1を作る。
+
##LB-Amp pre-cultured Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908)) tube 1 (10mL) was mixed with LB-Amp-agar(50&deg;C)(10ml)to produce sender containing bacterialplate-1.
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##LB-Ampで12hプレ培養したSender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](BW))の培養液-2(100&mu;l)をLB-Amp(9.9ml)に混ぜ、100倍希釈。この菌液10mlとLB-Amp-agar(50&deg;C)(10ml)を混ぜて、Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](BW))入りの固体培地-2を作る。
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##LB-Amp pre-cultured Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908)) tube 2(100&mu;l)was mixed with LB-Amp(9.9ml) and diluted 100-fold. 10ml of this solution was mixed with LB-Amp-agar(50&deg;C)(10ml) and created Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908))containing bacterial plate-2.
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##LB-Ampで12hプレ培養したSenderの培養液-2(10&mu;l)をLB-Amp(9.99ml)に混ぜ、1000倍希釈。この菌液10mlとLB-Amp-agar(50&deg;C)(10ml)を混ぜて、Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](BW))入りの固体培地-3を作る。
+
##LB-Amp pre-cultured Sender solution-2(10&mu;l) and LB-Amp(9.99ml)was mixed to dilute1000-fold.10ml of this solution and LB-Amp-agar(50&deg;C)(10ml) was mixed to create Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908) containing bacterial plate-3
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##ネガティブコントロール用としてLB-Amp(10ml)と、LB-Amp-agar(10ml)を混ぜ、固体培地を作った。
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#Lifted with nitrocellulose
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#ニトロセルロースでリフト
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##Receiver([http://partsregistry.org/Part:BBa_T9002 BBa_T9002](JW1908))colony was transfered to a nitrocellulose filter and placed on each of Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908))containing bacterial plate (1~3) and Sender-absent negative control plate (t=0). Determined the time required for the colonies to fluoresce depending on the bacterial concentration (100 and 1000-fold dilution).
-
##Receiver([http://partsregistry.org/Part:BBa_T9002 BBa_T9002](BW))のcolonyをニトロセルロースフィルターに写し取り、Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](BW))が入った固体培地(1~3)と、Senderなし(ネガティブコントロール用)の固体培地の上に置き(t=0)、菌の濃度(100倍,1000倍希釈)によってcolonyが光りだすまでの時間がどう変化するか調べた。
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#Method to detect fluorescence
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#GFPの確認方法
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##Plates cultured at 37&deg;C were exposed to UV (312nm) light once every 30 minutes to observe GFP fluorescence.
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##37&deg;Cで培養しているプレートを30分ごとにUV(312nm)を当てて、GFPが見えるか確認した。
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<BR>
<BR>
-
香取
 
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===method===
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===Testing different receivers-methods===
 +
#Receiver&sender pre-culture
 +
##Used Receivers were:
 +
###*[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]:ptet-luxR-plux-GFP(high copy)
 +
###*ptet-luxR-(low copy),[http://partsregistry.org/Part:BBa_J37032 BBa_J37032]:plux-GFP(high copy)
 +
###*[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]:ptet-luxR-plux-GFP(low copy)
 +
###*ptet-mLuxR(too sensitive)-plux-GFP
 +
###*ptet-luxR-plux-GFP-plac-aiiA 
 +
###*(all JW1908)Each was cultured in 2ml LB (37&deg;C,12h) and plated so that about 1000 colonies of receiver cells will grow.
 +
##Sender:[http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908) was cultured in 10mL LB in 50mL centrifuge tubes (37&deg;C,12h)
 +
#sender wash
 +
##Each receiver-containing medium was centrifuged in 50mL tubes at de20&deg;C, 3600rpm for 6min and supernatant discarded.
 +
##Added 10mL LB to each tube.
 +
##Repeated wash twice.
 +
#Creating bacterial plates
 +
##LB pre-cultured Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908)) tube 1 (10mL) was mixed with LB-agar(50&deg;C)(10ml)to produce sender containing bacterial plate-1.
 +
##LB pre-cultured Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908)) tube 2(100&mu;l)was mixed with LB(9.9ml) and diluted 100-fold. 10ml of this solution was mixed with LB-agar(50&deg;C)(10ml) and created Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908)) containing bacterial plate-2.
 +
##LB pre-cultured Sender solution-2(10&mu;l) and LB(9.99ml) was mixed to dilute 1000-fold.10ml of this solution and LB-agar(50&deg;C)(10ml) was mixed to create Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908)) containing bacterial plate-3
 +
#Lifted with nitrocellulose
 +
##Each Receiver colony was transfered to a nitrocellulose filter and placed on a Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908)) containing bacterial plate (1~3) or a sender-absent negative control plate(t=0) to observe how receiver type affects the time taken for the colonies to display visible fluorescence.
 +
#Method to detect fluorescence
 +
##Plates cultured at 37&deg;C were exposed to UV (312nm) light once every 30 minutes to observe GFP fluorescence.
<BR>
<BR>
-
===result-菌数ふり実験===
 
 +
===Varying bacterial numbers-results and discussion===
 +
===results===
-
 
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No Dilution
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希釈なし
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[[Image:Team-Chiba-IMG_0322-1.JPG|210px]]
[[Image:Team-Chiba-IMG_0322-1.JPG|210px]]
[[Image:Team-Chiba-IMG_0331-1.JPG|225px]]
[[Image:Team-Chiba-IMG_0331-1.JPG|225px]]
[[Image:Team-Chiba-IMG_0340.JPG|198px]]
[[Image:Team-Chiba-IMG_0340.JPG|198px]]
-
                    0h                          0.5h                         1.5h
+
                 0h                         0.5h                       1.0h
<BR>
<BR>
-
100倍希釈
+
100-fold dilution
[[Image:Team-Chiba-IMG_0322-100.JPG|210px]]
[[Image:Team-Chiba-IMG_0322-100.JPG|210px]]
[[Image:Team-Chiba-IMG_0331.JPG|200px]]
[[Image:Team-Chiba-IMG_0331.JPG|200px]]
[[Image:Team-Chiba-IMG_0340-100.JPG|212px]]
[[Image:Team-Chiba-IMG_0340-100.JPG|212px]]
[[Image:Team-Chiba-IMG_0349-100.JPG|179px]]
[[Image:Team-Chiba-IMG_0349-100.JPG|179px]]
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<BR>  
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<BR>            
-
                  0h                          0.5h                        1.5h                         2.0h
+
             0h                      0.5h                  1.0h                      1.5h
<BR>
<BR>
-
1000倍希釈
+
1000-fold dilution
[[Image:Team-Chiba-IMG_0322-1000.JPG|160px]]
[[Image:Team-Chiba-IMG_0322-1000.JPG|160px]]
[[Image:Team-Chiba-IMG_0331-1000.JPG|170px]]
[[Image:Team-Chiba-IMG_0331-1000.JPG|170px]]
Line 118: Line 138:
[[Image:Team-Chiba-IMG_0378-1000.JPG|200px]]
[[Image:Team-Chiba-IMG_0378-1000.JPG|200px]]
-
                0h                     0.5h                 1.5h               2.0h               2.5h
+
                  0h                    0.5h               1.0h           1.5h               2.0h
-
===result-いろんなレシーバー実験===
+
===discussion===
 +
We demonstrated that the GFP expression switch is delayed by the ratio of sender to receiver.
 +
The result indicates that the amount of AHL from one bacterium per time is constant and independent of bacteria number density.
 +
This is probaly because the sender has no feedback circuit of AHL production.
 +
Although this strategy can not change the time interval, we can manage the switch timing by changing the ratio of sender to receiver.
 +
 +
===Testing different receivers-results and discussion===
 +
 +
===results===
[[Image:Team-Chiba-IMG_0299.JPG.jpg|160px]]
[[Image:Team-Chiba-IMG_0299.JPG.jpg|160px]]
-
[[Image:Team-Chiba-IMG_0306-.jpg|175px]]
+
[[Image:Team-Chiba-IMG_0306-.jpg|178px]]
-
[[Image:Team-Chiba-IMG_0314-.jpg|160px]]
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[[Image:Team-Chiba-IMG_0314-.jpg|151px]]
-
[[Image:Team-Chiba-IMG_0319-.jpg|160px]]
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[[Image:Team-Chiba-IMG_0319-.jpg|154px]]
-
          0h                   0.5h                       1.0h                   1.5h
+
        0h                 0.5h                 1.0h               1.5h
-
1=N.C  
+
1=N.C
-
2=[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]:ptet-luxR-plux-GFP(high copy)
+
2=[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]:[https://2008.igem.org/Team:Chiba/Experiments:copy_number ptet-luxR-plux-GFP(high copy)]
-
3=ptet-luxR-(low copy),[http://partsregistry.org/Part:BBa_J37032 BBa_J37032]:plux-GFP(high copy)
+
3=[https://2008.igem.org/Team:Chiba/Experiments:copy_number ptet-luxR-(low copy)],[http://partsregistry.org/Part:BBa_J37032 BBa_J37032]:[https://2008.igem.org/Team:Chiba/Experiments:copy_number plux-GFP(high copy)]
-
4=[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]:ptet-luxR-plux-GFP(low copy)  
+
4=[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]:[https://2008.igem.org/Team:Chiba/Experiments:copy_number ptet-luxR-plux-GFP(low copy)]
-
5=ptet-mLuxR(too sensitive)-plux-GFP  
+
5=[https://2008.igem.org/Team:Chiba/Experiments:LuxR_mutant ptet-mLuxR(too sensitive)-plux-GFP]
 +
 
 +
6=N.C
 +
 
 +
7=[https://2008.igem.org/Team:Chiba/Project/Experiments:Signal_Molecule_Quencher ptet-luxR-plux-GFP-plac-aiiA]
 +
 
 +
===discussion===
 +
*Number 2,3 and 5 fluoresces in 30 min. We could not see time difference of these, it may be resulted from excess amount of sender bacteria.
 +
*Precise experiments controlling the number of sender are necessary for further discussion.
 +
*In comparizon with number 2 (T9002:high copy), there was no change of fluorescence intensity of number 4 (T9002:low copy) in 4 hours.
 +
*It is probably because of circuit working, since the AHL is provided enough for the receiver.
 +
 
 +
== Demo Experiment ~Temporal imaging system~ ==
 +
===Method===
 +
#Receiver(T9002)
 +
##Receiver:[http://partsregistry.org/Part:BBa_T9002 BBa_T9002], T9002-p15A and AiiA Receiver (JW1908) was cultured in 2mL LB-Amp-Cm, respectively. (37&deg;C,12h)
 +
##Pre-incubated Receivers was spotted so as to produce about 300 colonies on a nitrocellulose Filtter.
 +
##Incubated 37&deg;C,12h.
 +
#Sender(S03623)
 +
##Sender:[http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908) was cultured in 10mL of LB-Amp-Cm (37&deg;C,12h) and after 12 h, added 10ml of LB Ager-Amp-Cm into the incubated Sender.
 +
#Lifted Receiver colony with nitrocellulose Filter to the bacterial plate.
 +
#Cultured at 37&deg;C, 12h.
 +
#Exposed to UV (312nm) light once every 30 minutes to observe GFP fluorescence.
 +
 
 +
===Result===
 +
[[Image:Demo-flower Chiba.gif|frame|left|'''Pic. 1''' Flower should have blossomed!!!<br>
 +
Leaves, a stem and a flower was drawned with [http://partsregistry.org/Part:BBa_T9002 BBa_T9002], T9002-p15A, and AiiA Receiver, respectively on the plate containing [http://partsregistry.org/Part:BBa_S03623 BBa_S03623 (AHL sender)]]]
 +
<br clear=all>
 +
 
 +
===Discussion===
 +
GFP coloration of AiiA Receiver(flower blossom) wasn't observed.<br>
 +
We will try again with different method or in different condition until recognizing that the flower grow up!
-
6=N.C
 
-
7=ptet-luxR-plux-GFP-plac-aiiA
 
'''>[[Team:Chiba/Project#Demo Experiments|Back to the project page]]'''
'''>[[Team:Chiba/Project#Demo Experiments|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;" cellpadding="3" cellspacing="3" border="0" width="100%" align="center" class="menu" |
!align="center"|[[Team:Chiba|Home]]
!align="center"|[[Team:Chiba|Home]]
!align="center"|[[Team:Chiba/Team|The Team]]
!align="center"|[[Team:Chiba/Team|The Team]]
!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 12:44, 16 December 2008

Chiba-U.gif

Demonstration Experiment ~Senders~

Method

  1. Pre-culture
    1. Picked and cultured the following glycerol stocks in 2mL of LB:
      1. LB-Amp, [http://partsregistry.org/Part:BBa_T9002 BBa_T9002], (JW1908)
      2. LB-Amp+0.2%Glu, [http://partsregistry.org/Part:BBa_K084012 BBa_K084012]([http://partsregistry.org/Part:BBa_J04500 plac+rbs]+[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0161 LuxI(no LVA)]), (XL10G)
      3. LB-Amp+0.2%Glu, [http://partsregistry.org/Part:BBa_K084007 BBa_K084007]([http://partsregistry.org/Part:BBa_J04500 plac+rbs]+[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0178 LasI(no LVA)]), (XL10G)
    2. Cultured at 37°C for 12h.
  2. Culture
    1. Added 6.25% each of the pre-cultures to new LB medium.
      1. LB-Amp, [http://partsregistry.org/Part:BBa_T9002 BBa_T9002]
      2. LB-Amp+0.2%Glu, [http://partsregistry.org/Part:BBa_K084012 BBa_K084012]([http://partsregistry.org/Part:BBa_J04500 plac+rbs]+[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0161 LuxI(no LVA)]), [http://partsregistry.org/Part:BBa_K084007 BBa_K084007]([http://partsregistry.org/Part:BBa_J04500 plac+rbs]+[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0178 LasI(no LVA)])
    2. Cultured at 37°C for 4~5h。
  3. Wash
    1. Transfer 10mL each of the culture to 50mL centrifuge tubes.
    2. Centrifuged for 6min at 3600rpm,20°C and discarded the supernatant.
    3. Added LB-Amp to each centrifuge tube:
      1. 10mL to the tube that contains [http://partsregistry.org/Part:BBa_T9002 BBa_T9002]
      2. 5mL to the tube that contains [http://partsregistry.org/Part:BBa_K084012 BBa_K084012], [http://partsregistry.org/Part:BBa_K084007 BBa_K084007]
    4. Centrifuged for 6min, 3600rpm at 20°C the tube containing [http://partsregistry.org/Part:BBa_K084012 BBa_K084012], [http://partsregistry.org/Part:BBa_K084007 BBa_K084007] and discarded the supernatant.
    5. 10mL to the tube that contains [http://partsregistry.org/Part:BBa_K084012 BBa_K084012], [http://partsregistry.org/Part:BBa_K084007 BBa_K084007]
    6. Centrifuged for 6min, 3600rpm at 20°C the tube containing [http://partsregistry.org/Part:BBa_K084012 BBa_K084012], [http://partsregistry.org/Part:BBa_K084007 BBa_K084007] and discarded the supernatant.
    7. 5mL to the tube that contains [http://partsregistry.org/Part:BBa_K084012 BBa_K084012], [http://partsregistry.org/Part:BBa_K084007 BBa_K084007]
  4. Mix
    1. Mixed the sender cells [http://partsregistry.org/Part:BBa_K084012 BBa_K084012] and [http://partsregistry.org/Part:BBa_K084007 BBa_K084007] both with [http://partsregistry.org/Part:BBa_T9002 BBa_T9002] at a 1:1 ratio.
    2. Added 100μL each to a 96-well shallow plate (as shown in the figure).
      1. Green part is [http://partsregistry.org/Part:BBa_K084012 BBa_K084012]:[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]=1:1
      2. Red part is [http://partsregistry.org/Part:BBa_K084007 BBa_K084007]:[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]=1:1
      3. Uncolored part is [http://partsregistry.org/Part:BBa_T9002 BBa_T9002] alone.
  5. Culture and observe results

Results

Team-Chiba-demo-mihon.gif Green region: sender=LuxI, Red circular region: sender=Las I.


LuxI GFP is detected at 4h following mixing while LasI GFP is detected after 8h, thus successfully demonstrating time-delay depending on the sender used.


--Yoshimi 13:41, 29 October 2008 (UTC)

Demonstration Experiment ~Receivers~

Varying bacterial numbers: method

  1. Receiver(T9002) pre-incubation
    1. Receiver:[http://partsregistry.org/Part:BBa_T9002 BBa_T9002](JW1908)was cultured in 2mL LB-Amp. (37°C,12h)
    2. Pre-incubated Receiver([http://partsregistry.org/Part:BBa_T9002 BBa_T9002](JW1908))was plated so as to produce about 1000 colonies.
  2. Sender(S03623) pre-incubation
    1. Sender:[http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908) was cultured in 50mL entrifuge tubes in 10mL of LB-Amp (37°C,12h)(2tubes)
  3. Sender Wash
    1. Centrifuged 2 tubes containing([http://partsregistry.org/Part:BBa_T9002 BBa_T9002](JW1908))at 20°C,3600rpm for 6min and discarded supernatant.
    2. Added 10mL LB-Amp to each tube.
    3. Repeated wash twice.
  4. Creating bacterial plates
    1. LB-Amp pre-cultured Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908)) tube 1 (10mL) was mixed with LB-Amp-agar(50°C)(10ml)to produce sender containing bacterialplate-1.
    2. LB-Amp pre-cultured Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908)) tube 2(100μl)was mixed with LB-Amp(9.9ml) and diluted 100-fold. 10ml of this solution was mixed with LB-Amp-agar(50°C)(10ml) and created Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908))containing bacterial plate-2.
    3. LB-Amp pre-cultured Sender solution-2(10μl) and LB-Amp(9.99ml)was mixed to dilute1000-fold.10ml of this solution and LB-Amp-agar(50°C)(10ml) was mixed to create Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908) containing bacterial plate-3
  5. Lifted with nitrocellulose
    1. Receiver([http://partsregistry.org/Part:BBa_T9002 BBa_T9002](JW1908))colony was transfered to a nitrocellulose filter and placed on each of Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908))containing bacterial plate (1~3) and Sender-absent negative control plate (t=0). Determined the time required for the colonies to fluoresce depending on the bacterial concentration (100 and 1000-fold dilution).
  6. Method to detect fluorescence
    1. Plates cultured at 37°C were exposed to UV (312nm) light once every 30 minutes to observe GFP fluorescence.



Testing different receivers-methods

  1. Receiver&sender pre-culture
    1. Used Receivers were:
        • [http://partsregistry.org/Part:BBa_T9002 BBa_T9002]:ptet-luxR-plux-GFP(high copy)
        • ptet-luxR-(low copy),[http://partsregistry.org/Part:BBa_J37032 BBa_J37032]:plux-GFP(high copy)
        • [http://partsregistry.org/Part:BBa_T9002 BBa_T9002]:ptet-luxR-plux-GFP(low copy)
        • ptet-mLuxR(too sensitive)-plux-GFP
        • ptet-luxR-plux-GFP-plac-aiiA
        • (all JW1908)Each was cultured in 2ml LB (37°C,12h) and plated so that about 1000 colonies of receiver cells will grow.
    2. Sender:[http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908) was cultured in 10mL LB in 50mL centrifuge tubes (37°C,12h)
  2. sender wash
    1. Each receiver-containing medium was centrifuged in 50mL tubes at de20°C, 3600rpm for 6min and supernatant discarded.
    2. Added 10mL LB to each tube.
    3. Repeated wash twice.
  3. Creating bacterial plates
    1. LB pre-cultured Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908)) tube 1 (10mL) was mixed with LB-agar(50°C)(10ml)to produce sender containing bacterial plate-1.
    2. LB pre-cultured Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908)) tube 2(100μl)was mixed with LB(9.9ml) and diluted 100-fold. 10ml of this solution was mixed with LB-agar(50°C)(10ml) and created Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908)) containing bacterial plate-2.
    3. LB pre-cultured Sender solution-2(10μl) and LB(9.99ml) was mixed to dilute 1000-fold.10ml of this solution and LB-agar(50°C)(10ml) was mixed to create Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908)) containing bacterial plate-3
  4. Lifted with nitrocellulose
    1. Each Receiver colony was transfered to a nitrocellulose filter and placed on a Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908)) containing bacterial plate (1~3) or a sender-absent negative control plate(t=0) to observe how receiver type affects the time taken for the colonies to display visible fluorescence.
  5. Method to detect fluorescence
    1. Plates cultured at 37°C were exposed to UV (312nm) light once every 30 minutes to observe GFP fluorescence.


Varying bacterial numbers-results and discussion

results

No Dilution Team-Chiba-IMG 0322-1.JPG Team-Chiba-IMG 0331-1.JPG Team-Chiba-IMG 0340.JPG

                 0h                         0.5h                        1.0h



100-fold dilution Team-Chiba-IMG 0322-100.JPG Team-Chiba-IMG 0331.JPG Team-Chiba-IMG 0340-100.JPG Team-Chiba-IMG 0349-100.JPG

             0h                      0.5h                  1.0h                       1.5h


1000-fold dilution Team-Chiba-IMG 0322-1000.JPG Team-Chiba-IMG 0331-1000.JPG Team-Chiba-IMG 0341-1000.JPG Team-Chiba-IMG 0352-1000.JPG Team-Chiba-IMG 0378-1000.JPG

                 0h                    0.5h                1.0h            1.5h               2.0h

discussion

We demonstrated that the GFP expression switch is delayed by the ratio of sender to receiver. The result indicates that the amount of AHL from one bacterium per time is constant and independent of bacteria number density. This is probaly because the sender has no feedback circuit of AHL production. Although this strategy can not change the time interval, we can manage the switch timing by changing the ratio of sender to receiver.

Testing different receivers-results and discussion

results

Team-Chiba-IMG 0299.JPG.jpg Team-Chiba-IMG 0306-.jpg Team-Chiba-IMG 0314-.jpg Team-Chiba-IMG 0319-.jpg

        0h                 0.5h                 1.0h                1.5h

1=N.C

2=[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]:ptet-luxR-plux-GFP(high copy)

3=ptet-luxR-(low copy),[http://partsregistry.org/Part:BBa_J37032 BBa_J37032]:plux-GFP(high copy)

4=[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]:ptet-luxR-plux-GFP(low copy)

5=ptet-mLuxR(too sensitive)-plux-GFP

6=N.C

7=ptet-luxR-plux-GFP-plac-aiiA

discussion

  • Number 2,3 and 5 fluoresces in 30 min. We could not see time difference of these, it may be resulted from excess amount of sender bacteria.
  • Precise experiments controlling the number of sender are necessary for further discussion.
  • In comparizon with number 2 (T9002:high copy), there was no change of fluorescence intensity of number 4 (T9002:low copy) in 4 hours.
  • It is probably because of circuit working, since the AHL is provided enough for the receiver.

Demo Experiment ~Temporal imaging system~

Method

  1. Receiver(T9002)
    1. Receiver:[http://partsregistry.org/Part:BBa_T9002 BBa_T9002], T9002-p15A and AiiA Receiver (JW1908) was cultured in 2mL LB-Amp-Cm, respectively. (37°C,12h)
    2. Pre-incubated Receivers was spotted so as to produce about 300 colonies on a nitrocellulose Filtter.
    3. Incubated 37°C,12h.
  2. Sender(S03623)
    1. Sender:[http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908) was cultured in 10mL of LB-Amp-Cm (37°C,12h) and after 12 h, added 10ml of LB Ager-Amp-Cm into the incubated Sender.
  3. Lifted Receiver colony with nitrocellulose Filter to the bacterial plate.
  4. Cultured at 37°C, 12h.
  5. Exposed to UV (312nm) light once every 30 minutes to observe GFP fluorescence.

Result

Pic. 1 Flower should have blossomed!!!
Leaves, a stem and a flower was drawned with [http://partsregistry.org/Part:BBa_T9002 BBa_T9002], T9002-p15A, and AiiA Receiver, respectively on the plate containing [http://partsregistry.org/Part:BBa_S03623 BBa_S03623 (AHL sender)]


Discussion

GFP coloration of AiiA Receiver(flower blossom) wasn't observed.
We will try again with different method or in different condition until recognizing that the flower grow up!



>Back to the project page