Discussion on our protocols (Tom & Vincent)
- 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.
- 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
- 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.
- 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