Team:Chiba/Calendar-Home/23 October 2008

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[[Team:Chiba/Calendar-Home/22 October 2008|22 October 2008 <]]|[[Team:Chiba/Calendar-Home/24 October 2008|> 24 October 2008]]
[[Team:Chiba/Calendar-Home/22 October 2008|22 October 2008 <]]|[[Team:Chiba/Calendar-Home/24 October 2008|> 24 October 2008]]
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==Laboratory work==
===Team:Demo-Rs===
===Team:Demo-Rs===
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<BR>朝
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====Varying bacterial numbers====
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#Receiver(T9002) pre-incubation
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##Receiver:[http://partsregistry.org/Part:BBa_T9002 BBa_T9002](JW1908)wascultured in 2mL LB-Amp (37&deg;C,12h)
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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) pre-incubation
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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)(2 tubes)
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#Sender Wash
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##Centrifuged 2 tubes containing([http://partsregistry.org/Part:BBa_T9002 BBa_T9002](JW1908))at 20&deg;C,3600rpm for 6min and discarded supernatant.
 +
##Added 10mL LB-Amp to each tube.
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##Repeated wash twice.
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#Creating bacterial plates
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##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 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 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](JW1908) containing bacterial plate-3
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#Lifted with nitrocellulose
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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).
 +
#Method to detect fluorescence
 +
##Plates cultured at 37&deg;C were exposed to UV (312nm) light once every 30 minutes to observe GFP fluorescence.
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===[https://2008.igem.org/Team:Chiba/Demo_experiments:Receivers results]===
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===Testing different receivers===
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#Receiver&sender pre-culture
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##Used Receivers were:
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###*[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]:ptet-luxR-plux-GFP(high copy)
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###*ptet-luxR-(low copy),[http://partsregistry.org/Part:BBa_J37032 BBa_J37032]:plux-GFP(high copy)
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###*[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]:ptet-luxR-plux-GFP(low copy)
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###*ptet-mLuxR(too sensitive)-plux-GFP
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###*ptet-luxR-plux-GFP-plac-aiiA 
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###*（all JW1908）Each was cultured in 2ml LB (37&deg;C,12h) and plated so that about 1000 colonies of receiver cells will grow.
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##Sender:[http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908) was cultured in 10mL LB in 50mL centrifuge tubes (37&deg;C,12h)
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#sender wash
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##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
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##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
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##Plates cultured at 37&deg;C were exposed to UV (312nm) light once every 30 minutes to observe GFP fluorescence.
<BR>
<BR>
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sender(T9002) グリストをつついて10ml培養×3(LB-Amp)(①washなし、②washあり、③(100:1,1000:1)
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===[https://2008.igem.org/Team:Chiba/Demo_experiments:Receivers results]===
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<BR>
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receiver プレ培したものをコロニーが1000位になるようにプレートに撒く。
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夜
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　センダーが濁ったら②③はwash×3
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　　①は菌液10mlにLB-agar10mlを加える。（Ampも）
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　　②はwash後菌液10mlにLB-agarを10ml加える。（Ampも）
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　　③(1:100)はwashした菌液を0.1mlとLB-Ampを9.9mlまぜ、そこにLB-agarを10ml加える。
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　　　(1:1000)はwashした菌液を0.01mlとLB-Ampを9.99mlまぜ、そこにLB-agarを10ml加える。
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　　また、ネガコンようとしてLB10mlと、LB-agar10mlを混ぜてプレートにしたものを用意する。
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①②③、ネガコンをプレートに移して固まるまで待つ。固まったらreceiver(T9002)をプレートからニトロセルロースで写し取りそれぞれのプレートに2枚ずつはり、その時をスタートとして37℃の培養機の中にいれ、30分ごとにUVを当ててGFPが出ているかどうか確認する。
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Latest revision as of 02:05, 30 October 2008

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22 October 2008 <|> 24 October 2008

Contents

Laboratory work

Team:Demo-Rs

Varying bacterial numbers

  1. Receiver(T9002) pre-incubation
    1. Receiver:[http://partsregistry.org/Part:BBa_T9002 BBa_T9002](JW1908)wascultured 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)(2 tubes)
  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 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](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.

results

Testing different receivers

  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.


results

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