Team:Chiba/Demo experiments

From 2008.igem.org

(Difference between revisions)
(→Testing different receivers-methods)
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#Pre-culture
#Pre-culture
##Picked and cultured the following glycerol stocks in 2mL of LB:
##Picked and cultured the following glycerol stocks in 2mL of LB:
-
###LB-Amp, [http://partsregistry.org/Part:BBa_T9002 BBa_T9002], (BW)
+
###LB-Amp, [http://partsregistry.org/Part:BBa_T9002 BBa_T9002], (JW1908)
###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)
###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)
###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)
###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)
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===Varying bacterial numbers: method===
===Varying bacterial numbers: method===
#Receiver(T9002) pre-incubation
#Receiver(T9002) pre-incubation
-
##Receiver:[http://partsregistry.org/Part:BBa_T9002 BBa_T9002](BW)wascultured in 2mL LB-Amp (37°C,12h)
+
##Receiver:[http://partsregistry.org/Part:BBa_T9002 BBa_T9002](JW1908)wascultured in 2mL LB-Amp (37°C,12h)
-
##Pre-incubated Receiver([http://partsregistry.org/Part:BBa_T9002 BBa_T9002](BW))was plated so as to produce about 1000 colonies.
+
##Pre-incubated Receiver([http://partsregistry.org/Part:BBa_T9002 BBa_T9002](JW1908))was plated so as to produce about 1000 colonies.
#Sender(S03623) pre-incubation
#Sender(S03623) pre-incubation
-
##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)
+
##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)
#Sender Wash
#Sender Wash
-
##Centrifuged 2 tubes containing([http://partsregistry.org/Part:BBa_T9002 BBa_T9002](BW))at 20°C,3600rpm for 6min and discarded supernatant.
+
##Centrifuged 2 tubes containing([http://partsregistry.org/Part:BBa_T9002 BBa_T9002](JW1908))at 20°C,3600rpm for 6min and discarded supernatant.
##Added 10mL LB-Amp to each tube.
##Added 10mL LB-Amp to each tube.
##Repeated wash twice.
##Repeated wash twice.
#Creating bacterial plates
#Creating bacterial plates
-
##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.
+
##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.
-
##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.
+
##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.
-
##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
+
##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
#Lifted with nitrocellulose
#Lifted with nitrocellulose
-
##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).
+
##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).
#Method to detect fluorescence
#Method to detect fluorescence
##Plates cultured at 37°C were exposed to UV (312nm) light once every 30 minutes to observe GFP fluorescence.
##Plates cultured at 37°C were exposed to UV (312nm) light once every 30 minutes to observe GFP fluorescence.
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###*ptet-mLuxR(too sensitive)-plux-GFP
###*ptet-mLuxR(too sensitive)-plux-GFP
###*ptet-luxR-plux-GFP-plac-aiiA   
###*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.
+
###*（all JW1908）Each was cultured in 2ml LB (37°C,12h) and plated so that about 1000 colonies of receiver cells will grow.
-
##Sender:[http://partsregistry.org/Part:BBa_S03623 BBa_S03623](BW) was cultured in 10mL LB in 50mL centrifuge tubes (37°C,12h)
+
##Sender:[http://partsregistry.org/Part:BBa_S03623 BBa_S03623](JW1908) was cultured in 10mL LB in 50mL centrifuge tubes (37°C,12h)
#sender wash
#sender wash
##Each receiver-containing medium was centrifuged in 50mL tubes at de20°C, 3600rpm for 6min and supernatant discarded.
##Each receiver-containing medium was centrifuged in 50mL tubes at de20°C, 3600rpm for 6min and supernatant discarded.
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##Repeated wash twice.
##Repeated wash twice.
#Creating bacterial plates
#Creating bacterial plates
-
##LB pre-cultured Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](BW)) tube 1 (10mL) was mixed with LB-agar(50°C)(10ml)to produce sender containing bacterial plate-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.
-
##LB pre-cultured Sender([http://partsregistry.org/Part:BBa_S03623 BBa_S03623](BW)) 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](BW)) containing bacterial plate-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.  
-
##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](BW)) containing bacterial plate-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
#Lifted with nitrocellulose
#Lifted with nitrocellulose
-
##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.
+
##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.
#Method to detect fluorescence
#Method to detect fluorescence
##Plates cultured at 37°C were exposed to UV (312nm) light once every 30 minutes to observe GFP fluorescence.
##Plates cultured at 37°C were exposed to UV (312nm) light once every 30 minutes to observe GFP fluorescence.

Revision as of 20:19, 29 October 2008

Chiba-U.gif

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, BBa_T9002, (JW1908)
      2. LB-Amp+0.2%Glu, BBa_K084012(plac+rbs+LuxI(no LVA)), (XL10G)
      3. LB-Amp+0.2%Glu, BBa_K084007(plac+rbs+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, BBa_T9002
      2. LB-Amp+0.2%Glu, BBa_K084012(plac+rbs+LuxI(no LVA)), BBa_K084007(plac+rbs+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 BBa_T9002
      2. 5mL to the tube that contains BBa_K084012, BBa_K084007
    4. Centrifuged for 6min, 3600rpm at 20°C the tube containing BBa_K084012, BBa_K084007 and discarded　the supernatant.
    5. 10mL to the tube that contains BBa_K084012, BBa_K084007
    6. Centrifuged for 6min, 3600rpm at 20°C the tube containing BBa_K084012, BBa_K084007 and discarded　the supernatant.
    7. 5mL to the tube that contains BBa_K084012, BBa_K084007
  4. Mix
    1. Mixed the sender cells BBa_K084012 and BBa_K084007 both with 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 isBBa_K084012:BBa_T9002=1:1
      2. Red part is BBa_K084007:BBa_T9002=1:1
      3. Uncolored part is BBa_T9002 alone.
  5. Culture and observe results

Results

Team-Chiba-demo-mihon.gif 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:BBa_T9002(JW1908)wascultured in 2mL LB-Amp (37°C,12h)
    2. Pre-incubated Receiver(BBa_T9002(JW1908))was plated so as to produce about 1000 colonies.
  2. Sender(S03623) pre-incubation
    1. Sender: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(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(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(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(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(BBa_S03623(JW1908) containing bacterial plate-3
  5. Lifted with nitrocellulose
    1. Receiver(BBa_T9002(JW1908))colony was transfered to a nitrocellulose filter and placed on each of Sender(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:
        • BBa_T9002:ptet-luxR-plux-GFP(high copy)
        • ptet-luxR-(low copy),BBa_J37032:plux-GFP(high copy)
        • 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: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(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(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(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(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(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 Team-Chiba-IMG 0322-1.JPG Team-Chiba-IMG 0331-1.JPG Team-Chiba-IMG 0340.JPG 

                　0h                    　    0.5h                        1.0h



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　 　　　　　　　　　　　　　　　　1.0h                       1.5h


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                1.0h　　　　　　　　　   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

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=BBa_T9002:ptet-luxR-plux-GFP(high copy)

3=ptet-luxR-(low copy),BBa_J37032:plux-GFP(high copy)

4=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

>Back to the project page

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