IGEM:IMPERIAL/2006/Protocols/T9002

Part T9002: Transfer Function Characterisation

Motivation

The motivation behind this test construct is to characterise the transfer function linking AHL input to GFP output.

This will allow us to construct a standard transfer function curve which will allow us to relate GFP output to an unknown AHL input, forming the basis of an AHL assay.

Materials & Equipment

• Equipment
  • Wallac Victor 3 Multi-Well Fluorimeter
  • 11 Ependorf Tubes
  • Gilson Pippettes
  • 37oC Shaker
  • T9002 Report Sheet

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

Protocol

• 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
• Add the following to 11 seperate ependorf tubes to start AHL incubation:

Sample (ul)	Stock Concentration	AHL (ul)	Final AHL Concentration
990	1000uM	10	10uM
990	100uM	10	1uM
990	10uM	10	100nM
990	5uM	10	50nM
990	1uM	10	10nM
990	500nM	10	5nM
990	100nM	10	1nM
990	50nM	10	0.5nM
990	10nM	10	0.1nM
990	1nM	10	0.01nM
1000	N/A	0	0nM

• Incubate the ependorfs 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.

Last Updated: Tom 12:03, 8 August 2006 (BST)

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?
• JS: Please can you write a protocol that will decouple other experiments with this one in case we want to assay an unknown concentration of AHL. Thanks. (i.e. take an unknown concentration of AHL of x amount into the T9002....measure flourescence...etc)

IGEM:IMPERIAL/Protocols/T9002_AHL_assay