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

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

• Specifications
• Design
• Modelling
• Implementation
• Testing/Validation

Predator Test Construct

• Inoculate a culture from a single bacterial colony in LB growth medium containing appropriate antibiotic.
• Incubate at 37oC for 15 hours in a shaker.
• Remove cultures from shaker and dilute by a factor of 10, by adding 1ml of culture into 9ml of fresh LB medium.
• Incubate at 37oC for 2 hours in a shaker - This returns cells to exponential phase
• 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):

Sample (ul)	Stock Concentration	AHL (ul)	Final AHL Concentration
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

• 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
  • Prepare an ice bucket containing a pre-chilled 96 well plate - The ice is used to stop cell activity, therefore its imporant the plate stays cold
  • 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 - As the cells aren't in the shaker, the vortex helps to airate the culture
  • 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