Team:Chiba/Demo experiments

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(result and discussion-いろんなレシーバー実験)
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[[Image:Chiba-U.gif]]
[[Image:Chiba-U.gif]]
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{| style="color:white;background-color:Maroon" cellpadding="3"
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align="center"
!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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== Demo Experiment ~Senders~ ==
== Demo Experiment ~Senders~ ==
===Method===
===Method===
-
#プレ培養
+
#Pre-culture
-
##以下のグリストを各培養液2mL中にPick。
+
##Picked and cultured the following glycerol stocks in 2mL of LB:
###LB-Amp, [http://partsregistry.org/Part:BBa_T9002 BBa_T9002], (BW)
###LB-Amp, [http://partsregistry.org/Part:BBa_T9002 BBa_T9002], (BW)
-
###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
-
###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)
+
BBa_K084012]([http://partsregistry.org/Part:BBa_J04500
-
##37&deg;Cでしんとう培養。12h
+
plac+rbs]+[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0161
-
#本培養
+
LuxI(no LVA)]), (XL10G)
-
##プレ培養液を6.25%ずつ各培養液に添加する。
+
###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)
 +
##Cultured at 37&deg;C for 12h.
 +
#Culture
 +
##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
-
##37&deg;Cでしんとう培養。4~5h。
+
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)])
 +
##Cultured at 37&deg;C for 4~5h。
#Wash
#Wash
-
##遠心管50mL中に10mLずつ培養液を移す。
+
##Transfer 10mL each of the culture to 50mL centrifuge tubes.
-
##20&deg;C,3600rpmで6min遠心後、上澄みを捨てる。
+
##Centrifuged for 6min at 3600rpm,20&deg;C and discarded the supernatant.
-
##各遠心管にLB-Ampを加える。
+
##Added LB-Amp to each centrifuge tube:
-
###[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]が入った各遠心管に10mL
+
###10mL to the tube that contains
-
###[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
+
[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]
-
##[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。
+
###5mL to the tube that contains
-
##[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
+
[http://partsregistry.org/Part:BBa_K084012 BBa_K084012],
-
##[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。
+
[http://partsregistry.org/Part:BBa_K084007 BBa_K084007]
-
##[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
+
##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.
 +
##10mL to the tube that contains
 +
[http://partsregistry.org/Part:BBa_K084012 BBa_K084012],
 +
[http://partsregistry.org/Part:BBa_K084007 BBa_K084007]
 +
##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.
 +
##5mL to the tube that contains
 +
[http://partsregistry.org/Part:BBa_K084012 BBa_K084012],
 +
[http://partsregistry.org/Part:BBa_K084007 BBa_K084007]
#Mix
#Mix
-
##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。
+
##Mixed the sender cells [http://partsregistry.org/Part:BBa_K084012
-
##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。
+
BBa_K084012] and [http://partsregistry.org/Part:BBa_K084007
-
###緑の部分に[http://partsregistry.org/Part:BBa_K084012 BBa_K084012]:[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]=1:1
+
BBa_K084007] both with [http://partsregistry.org/Part:BBa_T9002
-
###赤の部分に[http://partsregistry.org/Part:BBa_K084007 BBa_K084007]:[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]=1:1
+
BBa_T9002] at a 1:1 ratio.
-
###無地の部分に[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]のみ
+
##Added 100&mu;L each to a 96-well shallow plate (as shown in the figure).
-
#培養&観察
+
###Green part is[http://partsregistry.org/Part:BBa_K084012
 +
BBa_K084012]:[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]=1:1
 +
###Red part is [http://partsregistry.org/Part:BBa_K084007
 +
BBa_K084007]:[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]=1:1
 +
###Uncolored part is [http://partsregistry.org/Part:BBa_T9002 BBa_T9002] alone.
 +
#Culture and observe results
=== Results ===
=== Results ===
-
[[Image:Team-Chiba-demo-mihon.gif|200px]] Green region: sender=LuxI, Red circular region: sender=Las I.
+
[[Image:Team-Chiba-demo-mihon.gif|200px]] Green region: sender=LuxI,
 +
Red circular region: sender=Las I.
<gallery>
<gallery>
Line 50: Line 86:
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)
-
</gallery> 
+
</gallery>
-
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。
+
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.
-
(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)
Line 61: Line 98:
== Demo Experiment ~Receivers~ ==
== Demo Experiment ~Receivers~ ==
-
===method-菌数ふり実験===
+
===Varying bacterial numbers: method===
-
#Receiver(T9002)のプレ培養
+
#Receiver(T9002) pre-incubation
-
##Receiver:[http://partsregistry.org/Part:BBa_T9002 BBa_T9002](BW)をLB-Amp 2ml培養(37&deg;C,12h)
+
##Receiver:[http://partsregistry.org/Part:BBa_T9002 BBa_T9002](BW)was
-
##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。
+
cultured in 2mL LB-Amp (37&deg;C,12h)
-
#Sender(S03623)のプレ培養
+
##Pre-incubated Receiver([http://partsregistry.org/Part:BBa_T9002
-
##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)
+
BBa_T9002](BW))was plated so as to produce about 1000 colonies.
-
#senderのWash
+
#Sender(S03623) pre-incubation
-
##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。
+
##Sender:[http://partsregistry.org/Part:BBa_S03623 BBa_S03623](BW) was
-
##各遠心管にLB-Amp10mlを加える。
+
cultured in 50mL centrifuge tubes in 10mL of LB-Amp (37&deg;C,12h)(2
-
##同様の操作をあと2回行った。
+
tubes)
-
#固体プレート作り
+
#Sender Wash
-
##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。
+
##Centrifuged 2 tubes containing
-
##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。
+
([http://partsregistry.org/Part:BBa_T9002 BBa_T9002](BW))at 20&deg;C,
-
##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。
+
3600rpm for 6min and discarded supernatant.
-
##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。
+
##Added 10mL LB-Amp to each tube.
-
#ニトロセルロースでリフト
+
##Repeated wash twice.
-
##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。
+
#Creating bacterial plates
-
#GFPの確認方法
+
##LB-Amp pre-cultured Sender([http://partsregistry.org/Part:BBa_S03623
-
##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。
+
BBa_S03623](BW)) tube 1 (10mL) was mixed with
 +
LB-Amp-agar(50&deg;C)(10ml)to produce sender containing bacterial
 +
plate-1.
 +
##LB-Amp pre-cultured Sender([http://partsregistry.org/Part:BBa_S03623
 +
BBa_S03623](BW)) 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](BW))
 +
containing bacterial plate-2.
 +
##LB-Amp pre-cultured Sender solution-2(10&mu;l) and LB-Amp(9.99ml)
 +
was mixed to dilute 1000-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](BW))
 +
containing bacterial plate-3
 +
#Lifted with nitrocellulose
 +
##Receiver([http://partsregistry.org/Part:BBa_T9002 BBa_T9002](BW))
 +
colony was transfered to a nitrocellulose filter and placed on each of
 +
Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](BW))
 +
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).
 +
#Method to detect fluorescence
 +
##Plates cultured at 37&deg;C were exposed to UV (312nm) light once
 +
every 30 minutes to observe GFP fluorescence.
Line 86: Line 147:
香取
香取
-
===method-いろんなレシーバー実験===
+
===Testing different receivers-methods===
-
#Receiver&senderのプレ培養
+
#Receiver&sender pre-culture
-
##用いたReceiverは   
+
##Used Receivers were:
-
     ・[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]:ptet-luxR-plux-GFP(high copy)
+
[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)
+
・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)  
+
[http://partsregistry.org/Part:BBa_T9002
 +
BBa_T9002]:ptet-luxR-plux-GFP(low copy)
-
     ・ptet-mLuxR(too sensitive)-plux-GFP  
+
・ptet-mLuxR(too sensitive)-plux-GFP
-
+
-
     ・ptet-luxR-plux-GFP-plac-aiiA
+
-
   
+
-
       (すべてBW)をLB 2ml培養(37&deg;C,12h)プレ培養したそれぞれのReceiverをコロニーが1000個くらいになるようにプレートにまく。
+
-
##Sender:[http://partsregistry.org/Part:BBa_S03623 BBa_S03623](BW)をLB 50ml遠心管で10ml培養(37&deg;C,12h)
+
・ptet-luxR-plux-GFP-plac-aiiA
-
#senderのWash
+
-
##各receiverの培養液を50mL遠心管でde20&deg;C,3600rpmで6min遠心後、上澄みを捨てる。
+
-
##各遠心管にLB10mlを加える。
+
-
##同様の操作をあと2回行った。
+
-
#固体プレート作り
+
-
##LB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-agar(50&deg;C)(10ml)を混ぜてsender入りの固体培地-1を作る。
+
-
##LB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(9.9ml)に混ぜ、100倍希釈。この菌液10mlとLBagar(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。
+
-
##LBで12hプレ培養したSenderの培養液-2(10&mu;l)をLB(9.99ml)に混ぜ、1000倍希釈。この菌液10mlとLB-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。
+
-
#ニトロセルロースでリフト
+
-
##各Receiver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)receiverの種類によってcolonyが光りだすまでの時間がどう変化するか調べた。
+
-
#GFPの確認方法
+
-
##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。
+
-
<BR>
+
-
===result and discussion-菌数ふり実験===
+
(all BW)
 +
              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](BW) 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-Amp pre-cultured Sender([http://partsregistry.org/Part:BBa_S03623
 +
BBa_S03623](BW)) tube 1 (10mL) was mixed with
 +
LB-Amp-agar(50&deg;C)(10ml)to produce sender containing bacterial
 +
plate-1.
 +
##LB-Amp pre-cultured Sender([http://partsregistry.org/Part:BBa_S03623
 +
BBa_S03623](BW)) 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](BW))
 +
containing bacterial plate-2.
 +
##LB-Amp pre-cultured Sender solution-2(10&mu;l) and LB-Amp(9.99ml)
 +
was mixed to dilute 1000-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](BW))
 +
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](BW)) 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>
 +
 +
===Varying bacterial numbers-results===
-
希釈なし
+
No Dilution
[[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]]
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100-fold dilution
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1000倍希釈
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1000-fold dilution
[[Image:Team-Chiba-IMG_0322-1000.JPG|160px]]
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===result and discussion-いろんなレシーバー実験===
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===Testing different receivers-results===
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2=[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]:ptet-luxR-plux-GFP(high copy)
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'''>[[Team:Chiba/Project#Demo Experiments|Back to the project page]]'''
'''>[[Team:Chiba/Project#Demo Experiments|Back to the project page]]'''
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!align="center"|[[Team:Chiba|Home]]
!align="center"|[[Team:Chiba|Home]]
!align="center"|[[Team:Chiba/Team|The Team]]
!align="center"|[[Team:Chiba/Team|The Team]]

Revision as of 18:21, 29 October 2008

Chiba-U.gif

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

Contents

Demo 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], (BW)
      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)

      1. 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)

    1. Cultured at 37°C for 12h.
  1. 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)])

    1. Cultured at 37°C for 4~5h。
  1. 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]

      1. 5mL to the tube that contains

[http://partsregistry.org/Part:BBa_K084012 BBa_K084012], [http://partsregistry.org/Part:BBa_K084007 BBa_K084007]

    1. 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.

    1. 10mL to the tube that contains

[http://partsregistry.org/Part:BBa_K084012 BBa_K084012], [http://partsregistry.org/Part:BBa_K084007 BBa_K084007]

    1. 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.

    1. 5mL to the tube that contains

[http://partsregistry.org/Part:BBa_K084012 BBa_K084012], [http://partsregistry.org/Part:BBa_K084007 BBa_K084007]

  1. 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.

    1. 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

      1. Red part is [http://partsregistry.org/Part:BBa_K084007

BBa_K084007]:[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]=1:1

      1. Uncolored part is [http://partsregistry.org/Part:BBa_T9002 BBa_T9002] alone.
  1. 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)

Demo Experiment ~Receivers~

Varying bacterial numbers: method

  1. Receiver(T9002) pre-incubation
    1. Receiver:[http://partsregistry.org/Part:BBa_T9002 BBa_T9002](BW)was

cultured in 2mL LB-Amp (37°C,12h)

    1. Pre-incubated Receiver([http://partsregistry.org/Part:BBa_T9002

BBa_T9002](BW))was plated so as to produce about 1000 colonies.

  1. Sender(S03623) pre-incubation
    1. Sender:[http://partsregistry.org/Part:BBa_S03623 BBa_S03623](BW) was

cultured in 50mL centrifuge tubes in 10mL of LB-Amp (37°C,12h)(2 tubes)

  1. Sender Wash
    1. Centrifuged 2 tubes containing

([http://partsregistry.org/Part:BBa_T9002 BBa_T9002](BW))at 20°C, 3600rpm for 6min and discarded supernatant.

    1. Added 10mL LB-Amp to each tube.
    2. Repeated wash twice.
  1. Creating bacterial plates
    1. LB-Amp pre-cultured Sender([http://partsregistry.org/Part:BBa_S03623

BBa_S03623](BW)) tube 1 (10mL) was mixed with LB-Amp-agar(50°C)(10ml)to produce sender containing bacterial plate-1.

    1. LB-Amp pre-cultured Sender([http://partsregistry.org/Part:BBa_S03623

BBa_S03623](BW)) 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](BW)) containing bacterial plate-2.

    1. LB-Amp pre-cultured Sender solution-2(10μl) and LB-Amp(9.99ml)

was mixed to dilute 1000-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](BW)) containing bacterial plate-3

  1. Lifted with nitrocellulose
    1. Receiver([http://partsregistry.org/Part:BBa_T9002 BBa_T9002](BW))

colony was transfered to a nitrocellulose filter and placed on each of Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](BW)) 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).

  1. Method to detect fluorescence
    1. Plates cultured at 37°C were exposed to UV (312nm) light once

every 30 minutes to observe GFP fluorescence.



香取

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 BW)

              Each was cultured in 2ml LB (37°C,12h) and plated so that about

1000 colonies of receiver cells will grow.

    1. Sender:[http://partsregistry.org/Part:BBa_S03623 BBa_S03623](BW) was

cultured in 10mL LB in 50mL centrifuge tubes (37°C,12h)

  1. sender wash
    1. Each receiver-containing medium was centrifuged in 50mL tubes at

de20°C, 3600rpm for 6min and supernatant discarded.

    1. Added 10mL LB to each tube.
    2. Repeated wash twice.
  1. Creating bacterial plates
    1. LB-Amp pre-cultured Sender([http://partsregistry.org/Part:BBa_S03623

BBa_S03623](BW)) tube 1 (10mL) was mixed with LB-Amp-agar(50°C)(10ml)to produce sender containing bacterial plate-1.

    1. LB-Amp pre-cultured Sender([http://partsregistry.org/Part:BBa_S03623

BBa_S03623](BW)) 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](BW)) containing bacterial plate-2.

    1. LB-Amp pre-cultured Sender solution-2(10μl) and LB-Amp(9.99ml)

was mixed to dilute 1000-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](BW)) containing bacterial plate-3

  1. 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](BW)) 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.

  1. 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

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

                0h                        0.5h                        1.5h



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

1.5h 2.0h


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                1.5h
  2.0h               2.5h

Testing different receivers-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


>Back to the project page

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Home The Team The Project Parts Submitted to the Registry Notebook