IGEM:IMPERIAL/2006/Protocols/J37015-LuxI

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Contents

Part J37015 Alternative Protocol: Measuring LuxI

Motivation

The motivation behind this protocol is to directly assessing the concentration of LuxI in the prey cell using a downstream GFP gene as a measurement. This is done as an alternative to measuring AHL indirectly using the T9002 assay. The concentrations of LuxI and AHL have so far been assumed to be equal. This protocol will allow us to compare the behaviour of the two chemicals, and if indeed, our assumption proves to be correct, the measurement of LuxI protocol can completely replace the less accurate indirect AHL measurement protocol.

VR: Be careful, we never said that LuxI and AHL are to be equal. That would be a poor assumption as AHL is catalyzed by LuxI and AHL diffuses through the membrane. Therefore you can expect a saturation of LuxI within hours whereas AHL will accumulate into the medium for much longer. I am worried with this assay, as our modelling of T9002 and J37015 shows that these two parts are driven by the same parameters when it comes to assess LuxI. We should put more thinking into testing this part
Deepti: See the Talk Page please. Im not sure what needs to be done.

JS: Can we assume that the relationship between LuxI and AHL is linear?

Equipment & Materials

  • Equipment
    • Wallac Victor 3 Multi-Well Fluorimeter
    • Ependorf Tubes
    • Gilson Pippettes
    • 37[[:Category:{{{1}}}|{{{1}}}]] Shaker
    • Microfuge
  • Materials
    • E.coli Growth Medium w/Ampicilin
    • E.coli Culture Containing J37015
    • GFP Standard Solution

Protocol

Day 1

  • Innoculate a culture of J37015 from 10μL (from stored stock) into 2ml LB Amp.
  • Incubate overnight in the 37[[:Category:{{{1}}}|{{{1}}}]] shaker.

Day 2

  • Prewarm LB AMP to 37[[:Category:{{{1}}}|{{{1}}}]] by placing in the 37[[:Category:{{{1}}}|{{{1}}}]] waterbath.
  • Measure and record the OD of the culture at 600nm.
  • Innoculate a sample of J37015 from the overnight culture into fresh (prewarmed) LB AMP to bring OD back to 0.1, according to the fomula below:
  • Volume of Overnight Culture (X) = (0.1/OD600_1)*2ml
  • Volume of fresh Culture (Y) = 2-X (Overnight Culture)
  • Return LB AMP to 37[[:Category:{{{1}}}|{{{1}}}]] waterbath
  • Incubate new culture at 37[[:Category:{{{1}}}|{{{1}}}]] for two hours in the shaker.
  • After 2 hours, measure OD of the J37015 culture at 600nm. Record this second OD value.
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