Team:Chiba/Demo experiments:Receivers

From 2008.igem.org

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=discussion=
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*We demonstrated that the GFP expression switch is delayed by the ratio of sender to receiver.
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*菌数を振ることでGFP発現を遅らせることができた。このことから菌一匹が単位時間に生産するAHL量は
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*The result indicates that the amount of AHL from one bacterium per time is constant and independent of bacteria number density.
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菌密度(細胞外のAHL量)に関わらず一定であることがわかる。これはsenderが(AHLによる？)フィードバック
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*This is probaly because the sender has no feedback circuit of AHL production.
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機構を持ってないためと考えられる。
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*Although this strategy can not change the time interval, we can manage the switch timing by changing the ratio of sender to receiver.
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これを利用すれば各レシーバーのGFP発現までの時間を遅らせることができる。(異なるレシーバー間の発現の時間差を広げたり縮めたりすることはできない。)
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===Testing different receivers-results and discussion===
===Testing different receivers-results and discussion===

Revision as of 00:04, 30 October 2008

Contents

Demo Experiment ~Receivers~

Varying bacterial numbers: method

  1. Receiver(T9002) pre-incubation
    1. Receiver:BBa_T9002(JW1908)wascultured in 2mL LB-Amp (37°C,12h)
    2. Pre-incubated Receiver(BBa_T9002(JW1908))was plated so as to produce about 1000 colonies.
  2. Sender(S03623) pre-incubation
    1. Sender: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(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(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(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(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(BBa_S03623(JW1908) containing bacterial plate-3
  5. Lifted with nitrocellulose
    1. Receiver(BBa_T9002(JW1908))colony was transfered to a nitrocellulose filter and placed on each of Sender(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:
        • BBa_T9002:ptet-luxR-plux-GFP(high copy)
        • ptet-luxR-(low copy),BBa_J37032:plux-GFP(high copy)
        • 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: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(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(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(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(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(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=BBa_T9002:ptet-luxR-plux-GFP(high copy)

3=ptet-luxR-(low copy),BBa_J37032:plux-GFP(high copy)

4=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

  • 2,3,5は開始から30分以内で蛍光が確認された。差が生じなかったのはsenderの量が過剰だったためと考えられる。(希釈なし)

今後はこの条件に加え菌数を減らして時間差が見れるかどうかを確認すべきである。

  • 2のT9002(high copy)に対し4のT9002(low copy)は4時間後も蛍光強度にほとんど変化は見られなった。

AHL自体は過剰にあるので回路に問題があると考えられる。 >Back to the project page

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