Team:Chiba/protocol/phenotype/timedelay

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>[[Team:Chiba/Internal|team chiba Internal]]<Br>
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>[[Team:Chiba|return to notebook]]<Br>
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>[[Team:Chiba/jk|return to 実験ログ]]<Br>
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>[[Team:Chiba/protocol|return to protocols page]]<Br>
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== Time-delay check(冨永)==
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== Time-delay check==
=== Purpose===
=== Purpose===
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=== Equipments　and Materials ===
=== Equipments　and Materials ===
====Equipment====
====Equipment====
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*shaking incubator(37°C,30°C)
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*shaking incubator(37&deg;C,30&deg;C)
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**Innova 4200 Benchtop or Floor-Stackable Incubator Shaker(37°C)
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**Innova 4200 Benchtop or Floor-Stackable Incubator Shaker(37&deg;C)
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**Taitec BioShaker BR-33FM(30°C,200rpm)
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**Taitec BioShaker BR-33FM(30&deg;C,200rpm)
*46-well plate(deep well)
*46-well plate(deep well)
*Fluoroskan Ascent 2.5(program:Ascent Software Version 2.6)
*Fluoroskan Ascent 2.5(program:Ascent Software Version 2.6)
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====Materials====
====Materials====
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*AHL(100uM)
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*AHL(100&mu;M) solution
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*E.coli Culture Containing T9002
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*E.coli Culture Containing BBa_T9002
*E.coli Culture Containing plasmids you will testing
*E.coli Culture Containing plasmids you will testing
 +
: ex.)[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)])
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::[http://partsregistry.org/Part:BBa_K084008 BBa_K084008]([http://partsregistry.org/Part:BBa_J04500 plac+rbs]+[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0170 RhlI(no LVA)])
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*If the plasmids you will testing are regulated by IPTG,add IPTG to the reaction culteres when you start the measurement.
=== Protocol ===
=== Protocol ===
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====Culturing overnight (before the testing day):====
====Culturing overnight (before the testing day):====
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#Inoculate all cultures you will testing from gloycerol stocks into 2mL of LB-ampicillin liquid medium.
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#Inoculate all cultures you will testing from gloycerol stocks into 2 mL of LB-ampicillin liquid medium.
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#Also inoculate a culture containing T9002 into 2mL of LB-ampicillin liquid medium.
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#Also inoculate a culture containing BBa_T9002 into 2 mL of LB-ampicillin liquid medium.
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#Incubate all cultures with shaking at 37°C(O/N).
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#Incubate all cultures with shaking at 37&deg;C(O/N).
====Following day====
====Following day====
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*cultures containing T9002
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*cultures containing BBa_T9002
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#Inoculate a culture by adding 100μL of the cultures into 40mL of LB-ampicillin medium.(in a flask)
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#Inoculate a culture by adding 100 &mu;L of the cultures into 40mL of LB-ampicillin medium.(in a flask).
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#Incubate a culture for 6-8 hours with shaking at 37°C.
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#Incubate a culture for 6-8 hours with shaking at 37&deg;C.
#Wash
#Wash
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##Aliquote 10mL of the culture into 50mL four 50mL falcon tubes.
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##Aliquote 10mL of the culture into 50 mL four 50 mL falcon tubes.
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##Cultures are centrifuged at 3500rpm for 6 minutes.
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##Cultures are centrifuged at 3500 rpm for 6 minutes.
##Dinspense supernatant.
##Dinspense supernatant.
##Add 10mL of new LB-ampicillin medium and resuspense with pipetting.
##Add 10mL of new LB-ampicillin medium and resuspense with pipetting.
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*cultures containing plasmids you will testing
*cultures containing plasmids you will testing
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#Inoculate cultures by adding 12.5μL of the cultures into 5mL of LB-ampicillin medium.
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#Inoculate cultures by adding 12.5 &mu;L of the cultures into 5 mL of LB-ampicillin medium.
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#Incubate cultures for 6-8 hours with shaking at 37°C.
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#Incubate cultures for 6-8 hours with shaking at 37&deg;C.
#Wash
#Wash
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##Cultures are centrifuged at 3500rpm for 6 minutes.
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##Cultures are centrifuged at 3500　rpm for 6 minutes.
##Dinspense supernatant.
##Dinspense supernatant.
##Add 3mL of new LB-ampicillin medium and resuspense with pipetting.
##Add 3mL of new LB-ampicillin medium and resuspense with pipetting.
#repeat washing process twice.  
#repeat washing process twice.  
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#Cultures are centrifuged at 3500rpm for 6 minutes.
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#Cultures are centrifuged at 3500　rpm for 6 minutes.
#Dinspense supernatant.
#Dinspense supernatant.
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#Add 5mL of new LB-ampicillin medium and resuspense with pipetting.
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#Add 5　mL of new LB-ampicillin medium and resuspense with pipetting.
#aliquote cultures into a 48-deep well plate(deep well).
#aliquote cultures into a 48-deep well plate(deep well).
====Measurement====
====Measurement====
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*Mix senders and receiver as shown in Table below:
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*Mix senders culture and receiver culture(Prepare three replicate cultures).
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<table width="450" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
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ex.)sender culture:500&mu;L,receiver culture:500&mu;L
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<tr>
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::Positive control:receiver culture with 100&mu;M AHL solution.
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<td width="150">Senders culture(μL)</td><td>T9002 culture(μL)</td><td>Receiver cells/Sender cells</td>
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::Negative control:receiver culture without sender culture and AHL solution.
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</tr>
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#Incubate testing cultures with shaking at 37&deg;C.
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<tr>
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#After some intervals,aliqupte 100&mu;L of testing cultures into a 96-well plate(shallow well).
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<td>500</td><td>500</td><td>1</td>
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</tr>
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<tr>
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<td>100</td><td>1000</td><td>10</td>
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</tr>
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<tr>
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<td>10</td><td>1000</td><td>100</td>
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</tr>
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</table>
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three replicate cultures
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#Incubate testing cultures with shaking at 37°C.
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#After some intervals,aliqupte 100μL of testing cultures into a 96-well plate(shallow well).
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#Measure fluorescence intensity.  
#Measure fluorescence intensity.  
*conditions
*conditions
**shaking(before measurement):On time = 1min,Off time = 10 sec,
**shaking(before measurement):On time = 1min,Off time = 10 sec,
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**integration time = 1000ms
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**integration time = 1000 ms
**Beam width:Normal Beam
**Beam width:Normal Beam
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**Wavelength pair = 485nm(excitation) and 527nm(emission)
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**Wavelength pair = 485 nm(excitation) and 527 nm(emission)
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[[Team:Chiba/Project/Experiments:Sender_Crosstalk|return to Sender experiments details]]
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[[Team:Chiba/Sender_experiments|return to Sender experiments details]]
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Latest revision as of 08:56, 30 October 2008

>return to notebook
>return to protocols page

Contents

Time-delay check

Purpose

To Confirm that communication using non-endogenous molecules results in slower activation of receivers.

Equipments　and Materials

Equipment

  • shaking incubator(37°C,30°C)
    • Innova 4200 Benchtop or Floor-Stackable Incubator Shaker(37°C)
    • Taitec BioShaker BR-33FM(30°C,200rpm)
  • 46-well plate(deep well)
  • Fluoroskan Ascent 2.5(program:Ascent Software Version 2.6)
  • Beckman Allegratm X-12R Centrifuga(Beckman Coulter)

Materials

  • AHL(100μM) solution
  • E.coli Culture Containing BBa_T9002
  • E.coli Culture Containing plasmids you will testing
ex.)[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)])
[http://partsregistry.org/Part:BBa_K084008 BBa_K084008]([http://partsregistry.org/Part:BBa_J04500 plac+rbs]+[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0170 RhlI(no LVA)])
  • If the plasmids you will testing are regulated by IPTG,add IPTG to the reaction culteres when you start the measurement.

Protocol

Culturing overnight (before the testing day):

  1. Inoculate all cultures you will testing from gloycerol stocks into 2 mL of LB-ampicillin liquid medium.
  2. Also inoculate a culture containing BBa_T9002 into 2 mL of LB-ampicillin liquid medium.
  3. Incubate all cultures with shaking at 37°C(O/N).

Following day

  • cultures containing BBa_T9002
  1. Inoculate a culture by adding 100 μL of the cultures into 40mL of LB-ampicillin medium.(in a flask).
  2. Incubate a culture for 6-8 hours with shaking at 37°C.
  3. Wash
    1. Aliquote 10mL of the culture into 50 mL four 50 mL falcon tubes.
    2. Cultures are centrifuged at 3500 rpm for 6 minutes.
    3. Dinspense supernatant.
    4. Add 10mL of new LB-ampicillin medium and resuspense with pipetting.
  4. aliquote 1mL of the cultures into a 48-deep well plate(deep well).
  • cultures containing plasmids you will testing
  1. Inoculate cultures by adding 12.5 μL of the cultures into 5 mL of LB-ampicillin medium.
  2. Incubate cultures for 6-8 hours with shaking at 37°C.
  3. Wash
    1. Cultures are centrifuged at 3500　rpm for 6 minutes.
    2. Dinspense supernatant.
    3. Add 3mL of new LB-ampicillin medium and resuspense with pipetting.
  4. repeat washing process twice.
  5. Cultures are centrifuged at 3500　rpm for 6 minutes.
  6. Dinspense supernatant.
  7. Add 5　mL of new LB-ampicillin medium and resuspense with pipetting.
  8. aliquote cultures into a 48-deep well plate(deep well).

Measurement

  • Mix senders culture and receiver culture(Prepare three replicate cultures).

ex.)sender culture:500μL,receiver culture:500μL

Positive control:receiver culture with 100μM AHL solution.
Negative control:receiver culture without sender culture and AHL solution.
  1. Incubate testing cultures with shaking at 37°C.
  2. After some intervals,aliqupte 100μL of testing cultures into a 96-well plate(shallow well).
  3. Measure fluorescence intensity.
  • conditions
    • shaking(before measurement):On time = 1min,Off time = 10 sec,
    • integration time = 1000 ms
    • Beam width:Normal Beam
    • Wavelength pair = 485 nm(excitation) and 527 nm(emission)

return to Sender experiments details

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