Team:Chiba/Demo experiments

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===Testing different receivers-methods===
===Testing different receivers-methods===

Revision as of 18:22, 29 October 2008

Chiba-U.gif

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Home The Team The Project Parts Submitted to the Registry Reference Notebook Acknowledgements

Contents

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)

      1. 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)

    1. Cultured at 37°C for 12h.
  1. 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)])

    1. Cultured at 37°C for 4~5h。
  1. 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]

      1. 5mL to the tube that contains

[http://partsregistry.org/Part:BBa_K084012 BBa_K084012], [http://partsregistry.org/Part:BBa_K084007 BBa_K084007]

    1. 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. 10mL to the tube that contains

[http://partsregistry.org/Part:BBa_K084012 BBa_K084012], [http://partsregistry.org/Part:BBa_K084007 BBa_K084007]

    1. 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. 5mL to the tube that contains

[http://partsregistry.org/Part:BBa_K084012 BBa_K084012], [http://partsregistry.org/Part:BBa_K084007 BBa_K084007]

  1. 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.

    1. 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

      1. Red part is [http://partsregistry.org/Part:BBa_K084007

BBa_K084007]:[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]=1:1

      1. Uncolored part is [http://partsregistry.org/Part:BBa_T9002 BBa_T9002] alone.
  1. Culture and observe results

Results

Team-Chiba-demo-mihon.gif Green region: sender=LuxI, Red circular region: sender=Las I.


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)was

cultured in 2mL LB-Amp (37°C,12h)

    1. Pre-incubated Receiver([http://partsregistry.org/Part:BBa_T9002

BBa_T9002](BW))was plated so as to produce about 1000 colonies.

  1. Sender(S03623) pre-incubation
    1. Sender:[http://partsregistry.org/Part:BBa_S03623 BBa_S03623](BW) was

cultured in 50mL centrifuge tubes in 10mL of LB-Amp (37°C,12h)(2 tubes)

  1. 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.

    1. Added 10mL LB-Amp to each tube.
    2. Repeated wash twice.
  1. 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. 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. 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. 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).

  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.



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. 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. Added 10mL LB to each tube.
    2. Repeated wash twice.
  1. 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. 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. 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 Team-Chiba-IMG 0322-1.JPG Team-Chiba-IMG 0331-1.JPG Team-Chiba-IMG 0340.JPG

                0h                        0.5h                        1.5h



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

1.5h 2.0h


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                1.5h
  2.0h               2.5h

Testing different receivers-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=[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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