From 2008.igem.org

Revision as of 21:55, 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) >Back to the project page

• Recent changes
• What links here
• Related changes
• Special pages
• My preferences

• Printable version
• Permanent link
• Privacy policy
• Disclaimers