IGEM:IMPERIAL/2006/project/Oscillator/project browser/Test Sensing Predator Construct/TestingValidation: Difference between revisions

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!height="25pt" width="80pt"|Actual Part
!height="25pt" width="80pt"|Actual Part
!colspan="4"| Logo of the Part
!colspan="4"| [[Image:J37016 logo.png|300px]]
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!height="25pt" width="80pt"|Sub Parts
!height="25pt" width="80pt"|Sub Parts
! [[IGEM:IMPERIAL/2006/project/Oscillator/project browser/intermediate_part| intermediate_part]]
! <bbpart>R0062</bbpart>
! [[IGEM:IMPERIAL/2006/project/Oscillator/project browser/intermediate_part| intermediate_part]]
! <bbpart>B0034</bbpart>
! [[IGEM:IMPERIAL/2006/project/Oscillator/project browser/intermediate_part| intermediate_part]]
! <bbpart>C0062</bbpart>
! [[IGEM:IMPERIAL/2006/project/Oscillator/project browser/intermediate_part| intermediate_part]]
! <bbpart>I13504</bbpart>
|}
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==Motivations==


*Inoculate a culture from a single bacterial colony in LB growth medium containing appropriate antibiotic.
*Test GFP output for known concentrations of AHL and generate a trasfer curve.
*Incubate at 37oC for 15 hours in a shaker.
*GFP is in place of the AiiA gene.  
*Remove cultures from shaker and dilute by a factor of 10, by adding 1ml of culture into 9ml of fresh LB medium.
*We assume that GFP and AiiA have the same rate of transcription so the transfer curve can be use to characterize the sensing part of the predator.
*Incubate at 37oC for 2 hours in a shaker - '''<font color = green>This returns cells to exponential phase</font>'''
*We have a stock AHL solution of 1000uM
*Serial dilute to give final 1ml stock concentrations of 1000uM, 100uM, 10uM, 1uM, 100nM, 10nM, 1nM (check the -20oC Freezer as these may be already made up).
*Add the following to eight different labelled ependorf tubes (remember to add the AHL last):


<br>
==Protocols==
{| border="1" cellpadding="5"
!<u>Sample (ul)</u> || <u>Stock Concentration</u> !! <u>AHL (ul)</u> !! <u>Final AHL Concentration</u>
|-
|990
|1000uM
|10
|10uM
|-
|990
|100uM
|10
|1uM
|-
|990
|10uM
|10
|100nM
|-
|990
|1uM
|10
|10nM
|-
|990
|100nM
|10
|1nM
|-
|990
|10nM
|10
|0.1nM
|-
|990
|1nM
|10
|0.01nM
|-
|1000
|N/A
|0
|0nM
|}
<br>
*Add 200uL of growth medium to a well to act as a control
*Vortex tubes for 1 second
*Incubate the culture in a 37oC incubator, taking readings every 30minutes


*Taking readings
[http://openwetware.org/wiki/IGEM:IMPERIAL/2006/Protocols/T9002 Protocol for predator sensing test construct]
**Prepare an ice bucket containing a pre-chilled 96 well plate - '''<font color = green>The ice is used to stop cell activity, therefore its imporant the plate stays cold</font>'''
**Remove the tubes from the incubator
**Pippet 200ul samples from each tube into the 96 well plate inside the ice bucket
**Vortex the tubes for 1 second - '''<font color = green>As the cells aren't in the shaker, the vortex helps to airate the culture</font>'''
**Return tubes to the oven
**Repeat for a total of 4 measurments
*Take 96 well plate in ice bucket to the SAF
*Record data from the fluorimeter


References
==Results==


Tom Hinson
[http://openwetware.org/wiki/IGEM:Imperial/Results/J37016 Results for the predator sensing test construct]

Latest revision as of 15:40, 29 October 2006

Super Parts Predator Construct
Actual Part
Sub Parts <bbpart>R0062</bbpart> <bbpart>B0034</bbpart> <bbpart>C0062</bbpart> <bbpart>I13504</bbpart>


Motivations

  • Test GFP output for known concentrations of AHL and generate a trasfer curve.
  • GFP is in place of the AiiA gene.
  • We assume that GFP and AiiA have the same rate of transcription so the transfer curve can be use to characterize the sensing part of the predator.

Protocols

Protocol for predator sensing test construct

Results

Results for the predator sensing test construct

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