Team:Chiba/protocol/phenotype/timedelay

From 2008.igem.org

(Difference between revisions)
(→Measurement)
(→Materials)
 
(19 intermediate revisions not shown)
Line 1: Line 1:
<html><link rel="stylesheet" href="https://2008.igem.org/wiki/index.php?title=User:Hiroki/style.css&action=raw&ctype=text/css" type="text/css" /></html>
<html><link rel="stylesheet" href="https://2008.igem.org/wiki/index.php?title=User:Hiroki/style.css&action=raw&ctype=text/css" type="text/css" /></html>
-
>[[Team:Chiba/Internal|team chiba Internal]]<Br>
+
>[[Team:Chiba|return to notebook]]<Br>
-
>[[Team:Chiba/jk|return to 実験ログ]]<Br>
+
>[[Team:Chiba/protocol|return to protocols page]]<Br>
-
>[[Team:Chiba/protocol/j|return to protocols page]]<Br>
+
-
== Time-delay check(冨永)==
+
== Time-delay check==
=== Purpose===
=== Purpose===
Line 13: Line 12:
=== Equipments　and Materials ===
=== Equipments　and Materials ===
====Equipment====
====Equipment====
-
*shaking incubator(37°C,30°C)
+
*shaking incubator(37&deg;C,30&deg;C)
-
**Innova 4200 Benchtop or Floor-Stackable Incubator Shaker(37°C)
+
**Innova 4200 Benchtop or Floor-Stackable Incubator Shaker(37&deg;C)
-
**Taitec BioShaker BR-33FM(30°C,200rpm)
+
**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)
Line 21: Line 20:
====Materials====
====Materials====
-
*AHL(100uM)
+
*AHL(100&mu;M) solution
-
*E.coli Culture Containing T9002
+
*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)])
 +
::[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 ===
=== Protocol ===
Line 29: Line 31:
====Culturing overnight (before the testing day):====
====Culturing overnight (before the testing day):====
-
#Inoculate all cultures you will testing from gloycerol stocks into 2mL of LB-ampicillin liquid medium.
+
#Inoculate all cultures you will testing from gloycerol stocks into 2 mL of LB-ampicillin liquid medium.
-
#Also inoculate a culture containing T9002 into 2mL of LB-ampicillin liquid medium.
+
#Also inoculate a culture containing BBa_T9002 into 2 mL of LB-ampicillin liquid medium.
-
#Incubate all cultures with shaking at 37°C(O/N).
+
#Incubate all cultures with shaking at 37&deg;C(O/N).
====Following day====
====Following day====
-
*cultures containing T9002
+
*cultures containing BBa_T9002
-
#Inoculate a culture by adding 100μL of the cultures into 40mL of LB-ampicillin medium.(in a flask)
+
#Inoculate a culture by adding 100 &mu;L of the cultures into 40mL of LB-ampicillin medium.(in a flask).
-
#Incubate a culture for 6-8 hours with shaking at 37°C.
+
#Incubate a culture for 6-8 hours with shaking at 37&deg;C.
#Wash
#Wash
-
##Aliquote 10mL of the culture into 50mL four 50mL falcon tubes.
+
##Aliquote 10mL of the culture into 50 mL four 50 mL falcon tubes.
-
##Cultures are centrifuged at 3500rpm for 6 minutes.
+
##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.
Line 45: Line 47:
*cultures containing plasmids you will testing
*cultures containing plasmids you will testing
-
#Inoculate cultures by adding 12.5μL of the cultures into 5mL of LB-ampicillin medium.
+
#Inoculate cultures by adding 12.5 &mu;L of the cultures into 5 mL of LB-ampicillin medium.
-
#Incubate cultures for 6-8 hours with shaking at 37°C.
+
#Incubate cultures for 6-8 hours with shaking at 37&deg;C.
#Wash
#Wash
-
##Cultures are centrifuged at 3500rpm for 6 minutes.
+
##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.  
-
#Cultures are centrifuged at 3500rpm for 6 minutes.
+
#Cultures are centrifuged at 3500　rpm for 6 minutes.
#Dinspense supernatant.
#Dinspense supernatant.
-
#Add 5mL of new LB-ampicillin medium and resuspense with pipetting.
+
#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====
-
*Mix senders and receiver as shown in Table below:
+
*Mix senders culture and receiver culture(Prepare three replicate cultures).
-
<table width="450" border="4" cellpadding="0" cellspacing="0" bordercolor="#000000">
+
ex.)sender culture:500&mu;L,receiver culture:500&mu;L
-
<tr>
+
::Positive control:receiver culture with 100&mu;M AHL solution.
-
<td width="150">Senders culture(μL)</td><td>T9002 culture(μL)</td><td>Receiver cells/Sender cells</td>
+
::Negative control:receiver culture without sender culture and AHL solution.
-
</tr>
+
#Incubate testing cultures with shaking at 37&deg;C.
-
<tr>
+
#After some intervals,aliqupte 100&mu;L of testing cultures into a 96-well plate(shallow well).
-
<td>500</td><td>500</td><td>1</td>
+
-
</tr>
+
-
<tr>
+
-
<td>100</td><td>1000</td><td>10</td>
+
-
</tr>
+
-
<tr>
+
-
<td>10</td><td>1000</td><td>100</td>
+
-
</tr>
+
-
</table>
+
-
three replicate cultures
+
-
 
+
-
#Incubate testing cultures with shaking at 37°C.
+
-
#After some intervals,aliqupte 100μL of testing cultures into a 96-well plate(shallow well).
+
#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,
-
**integration time = 1000ms
+
**integration time = 1000 ms
**Beam width:Normal Beam
**Beam width:Normal Beam
-
**Wavelength pair = 485nm(excitation) and 527nm(emission)
+
**Wavelength pair = 485 nm(excitation) and 527 nm(emission)
[[Team:Chiba/Project/Experiments:Sender_Crosstalk|return to Sender experiments details]]
[[Team:Chiba/Project/Experiments:Sender_Crosstalk|return to Sender experiments details]]

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

Retrieved from "http://2008.igem.org/Team:Chiba/protocol/phenotype/timedelay"