IGEM:IMPERIAL/2006/ProjectCalendar/2006-8-17

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Contents

Discussion on our protocols (Tom & Vincent)

General Issues

  • Need to calibrate Victor 3 to get OD600=f(ABS_490)
    • grow a culture overnight
    • put the culture on ice for 1h
    • measure and record OD600
    • create a dilution series of culture (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7)
    • inoculate a 96 well plate with the dilution series, keep it on ice.
    • Use Victor 3 to read ABS_490
    • plot OD600 function of ABS_490
  • Need to create a fresh batch of T9002 and make of frozen stock of it.
  • Need to change AHL dilution series to fit better log scale.

T9002

  • Should use full 96 well plate: 8 repeats. New layout needed (8 repeats + standard GFP + blank medium) - Done!
    • means that we need to work-out new volumes needed for cultures and AHL inoculation
    • 1.5ml Eppendorf are too small, need bigger ones 2ml
      • TH: Sue has advised we don't have any 2ml Eppendorf tubes, would need to use the little white capped tubes
  • Need to make final decision on dilution method - Done!
  • To get pre-warmed media we should always store next volume needed in the incubator and not the full bottle. - Done!
  • We want to put AHL first into Eppendorf (before culture) ( can't remember why ?) - Done!
  • Check variability of Victor 3 measurements (multiple reading of the same plate) - Done!
  • Waterbath doesn't seem to be helpful (cells are dying). it is about doing only one measurement or finding an available 37C shacker. - Done!
  • Need to build a standard Excel sheet to process experimental data automaticaly:
    • convert ABS_490 -> OD600 - Done!
    • normalize GFP with OD600 - Done!
    • normalize GFP with standard GFP solution and remove background - Half-Done!
    • Compute Vmax and Km

J37016

  • Should benefit from all the improvements done on T9002. It is the same thing at the end of the day.
  • We assume that we will be at steady state after 4h.
  • 8 repeats + standard GFP + blank medium. Need standard layout.
  • Need Excel sheet to workout data processing + parameter extractions.

J37015

  • Need to validate that OD=0.1 helps to control positive feedback
    • diluting a culture of cells during a all day between OD=0.05 and 0.2
    • test GFP level at the end of the day to assess LuxI level
    • positive control with o/n culture
  • We would create a stock in the -3C fridge of this low density cell culture if it helps to avoid saturation.
  • To characterize we would then induce with AHL at min level of sensitivity of T9002
  • Need to set-up a protocol to assess GFP level in J37015 in time
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