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Revision as of 23:32, 29 October 2008

Demo Experiment ~Receivers~

Varying bacterial numbers: method

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.

Testing different receivers-methods

1. Receiver&sender pre-culture
   1. Used Receivers were:
      1. • [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

                　0h                    　    0.5h                        1.0h

100-fold dilution

    　　　　　　　　　　　　0h                      0.5h　 　　　　　　　　　　　1.0h                       1.5h

1000-fold dilution

   　　            0h                 　  0.5h                1.0h　　　　　　　1.5h               2.0h

discussion

• 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

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 機構を持ってないためと考えられる。 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。)

Testing different receivers-results and discussion

results

        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

• 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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