Team:Chiba/Demo experiments

From 2008.igem.org

Revision as of 18:34, 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], (BW)
      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.

1. 1. 10mL to the tube that contains [http://partsregistry.org/Part:BBa_K084012 BBa_K084012], [http://partsregistry.org/Part:BBa_K084007 BBa_K084007]
   2. 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.

1. 1. 5mL to the tube that contains [http://partsregistry.org/Part:BBa_K084012 BBa_K084012], [http://partsregistry.org/Part:BBa_K084007 BBa_K084007]
2. 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.
3. 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](BW)wascultured in 2mL LB-Amp (37°C,12h)
   2. Pre-incubated Receiver([http://partsregistry.org/Part:BBa_T9002 BBa_T9002](BW))was plated so as to produce about 1000 colonies.
2. Sender(S03623) pre-incubation
   1. Sender:[http://partsregistry.org/Part:BBa_S03623 BBa_S03623](BW) 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](BW))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](BW)) 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](BW)) 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](BW))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](BW)

containing bacterial plate-3

1. Lifted with nitrocellulose
   1. Receiver([http://partsregistry.org/Part:BBa_T9002 BBa_T9002](BW))colony was transfered to a nitrocellulose filter and placed on each of Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](BW))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).
2. 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:

・[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 BW）

              Each was cultured in 2ml LB (37°C,12h) and plated so that about

1000 colonies of receiver cells will grow.

1. 1. Sender:[http://partsregistry.org/Part:BBa_S03623 BBa_S03623](BW) was

cultured in 10mL LB in 50mL centrifuge tubes (37°C,12h)

1. sender wash
   1. Each receiver-containing medium was centrifuged in 50mL tubes at

de20°C, 3600rpm for 6min and supernatant discarded.

1. 1. Added 10mL LB to each tube.
   2. Repeated wash twice.
2. Creating bacterial plates
   1. LB-Amp pre-cultured Sender([http://partsregistry.org/Part:BBa_S03623

BBa_S03623](BW)) tube 1 (10mL) was mixed with LB-Amp-agar(50°C)(10ml)to produce sender containing bacterial plate-1.

1. 1. LB-Amp pre-cultured Sender([http://partsregistry.org/Part:BBa_S03623

BBa_S03623](BW)) 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](BW)) containing bacterial plate-2.

1. 1. 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](BW)) containing bacterial plate-3

1. 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](BW)) 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.

1. 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

No Dilution

                0h                        0.5h                        1.5h

100-fold dilution

                 0h                        0.5h

1.5h 2.0h

1000-fold dilution

               0h                   0.5h                1.5h
  2.0h               2.5h

Testing different receivers-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

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