IGEM:IMPERIAL/Protocols/T9002 AHL assay

AHL assay from T9002 characterization

UNFINISHED !!!!

Motivation

This protocols is designed to measure an unknown concentration of AHL in a medium.

Materials & Equipment

• Equipment
  • Wallac Victor 3 Multi-Well Fluorimeter
  • Ependorf Tubes
  • Gilson Pippettes
  • 37oC Shaker

• Materials

• • GFP Standard Solution
  • E.coli Growth Medium w/Ampicilin (LB/M9)
  • E.coli Culture Containing T9002

Protocol

Prepare T9002 cells:

• Inoculate a culture from 10ul of stored T9002 in 2ml LB/M9 growth medium containing 50ug/ml Ampicilin.
• Incubate at 37oC for overnight in a shaker.
• Measure and record OD
• Remove cultures from shaker and dilute so the culture is at an OD of 0.1 using 2ml of a prewarmed LB/M9 + Ampicilin.
  • measured_OD/0.1 x 2ml = Total volume required for dilution
• Incubate at 37oC for 2 hours in a shaker - This returns cells to exponential phase
• Measure and record the OD
• Dilute again with prewarmed LB/M9 for an OD of 0.1 using 15ml of a prewarmed LB/M9 + Ampicilin.
  • measured_OD/0.1 x 15ml = Total volume required for dilution

Prepare medium to assay:

• spin down solution and retreive supernatan
• Spin down T9002 solution and get rid of the supernatan to avoid dilution effect when measuring AHL.
• Resuspend T9002 cells into solution to be measured.

AHL incubation and GFP reading

• Incubate the ependorf in a 37oC shaker for 4 hours so GFP expression can reach steady state.
• After the 4 hours add 200uL samples from the ependorf tubes to the 96 well plate
• Do this for 5 repeats
• Add 200uL of growth medium to a well to act as a control
• Add 200uL of 200x diluted GFP standard solution
• Take 3 readings with a 20 minute interval
• Taking readings
  • Take the plate from the water bath
  • Use the Victor3 to measure flourescence and absorbance
  • Return the plate to the water bath

Any questions, see Tom.

Many thanks to Drew Endy and Barry Canton from MIT for providing the protocol on which this is based.

Potential Issues

• M9 or LB?
  • Need information from J37015 as to whether AHL production can be accomplished in M9
• What is the GFP standard solution? Do we know its concentration? How stable is that? half life?
• Growing all the five repeats together in one ependorf tube, can we get around this?
  • Incuabtor that can shake with the 96 well plate?