From 2008.igem.org

Revision as of 20:19, 29 October 2008

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], (JW1908)
      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)
      3. 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)
   2. Cultured at 37°C for 12h.
2. 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)])
   2. Cultured at 37°C for 4～5h。
3. 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]
      2. 5mL to the tube that contains [http://partsregistry.org/Part:BBa_K084012 BBa_K084012], [http://partsregistry.org/Part:BBa_K084007 BBa_K084007]
   4. 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.
   5. 10mL to the tube that contains [http://partsregistry.org/Part:BBa_K084012 BBa_K084012], [http://partsregistry.org/Part:BBa_K084007 BBa_K084007]
   6. 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.
   7. 5mL to the tube that contains [http://partsregistry.org/Part:BBa_K084012 BBa_K084012], [http://partsregistry.org/Part:BBa_K084007 BBa_K084007]
4. 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.
   2. 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
      2. Red part is [http://partsregistry.org/Part:BBa_K084007 BBa_K084007]:[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]=1:1
      3. Uncolored part is [http://partsregistry.org/Part:BBa_T9002 BBa_T9002] alone.
5. Culture and observe results

Results

Green region: sender=LuxI, Red circular region: sender=Las I.

0 h

4 h (Lux I GFP detected)

8 h (Lux I GFP and Las I GFP detected)

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](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)(2tubes)
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 dilute1000-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

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