IGEM:IMPERIAL/2006/Protocols/T9002
From OpenWetWare
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
- 12 Ependorf Tubes
- Gilson Pippettes
- 37oC Shaker
- T9002 Report Sheet
- Materials
- Dilution series of AHL [1nM, 5nM, 10nM, 50nM, 500nM, 100nM, 1mM] /* need better spread AHL level according to log scale*/
- GFP Standard Solution
- E.coli Growth Medium w/Ampicilin (LB/M9)
- E.coli DH5a Culture Containing T9002
Protocol
<showhide>
- 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. __HIDER__
<hide>
- This is to get a good stock of cells for use in the experiment. After the overnight culture the cells will be in stationary phase
</hide> </showhide> <showhide>
- Following day, prewarm LB/M9 to 37oC by placing in the 37oC incubator __HIDER__
<hide>
- Use the grey non-shaking incubator in the teaching lab. This is done now so that when you need to use the media later on it'll be prewarmed
</hide></showhide>
- Measure and record OD600_1 in report sheet
<showhide>
- Inoculate a 8ml fresh culture from the o/n to bring back the OD600 to 0.1, use prewarmed LB/M9 + Ampicilin. __HIDER__
<hide>
- Using prewarmed LB/M9 prevents a temperature shock to the culture, which would increase lag time
</hide> </showhide>
- Volume used to inoculate new culture = (0.1/OD600_1)*8ml
- Return LB/M9 to 37oC incubator
<showhide>
- Incubate new culture at 37oC for 2 hours in a shaker __HIDER__
<hide>
- This returns cells to exponential phase from stationary phase
</hide> </showhide>
- After the 2 hours measure and record the OD600_2 in report sheet
<showhide>
- Dilute again for an OD of 0.1 in a new culture of 15ml of a prewarmed LB/M9 + Ampicilin. __HIDER__
<hide>
- This dilution gives a standard OD to which to innoculate the culture with AHL (in this case 0.1). Innoculating at different ODs is known to give different results, so it is important a standard OD is used
</hide> </showhide>
- Volume used to inoculate new culture = (0.1/OD600_2)*15ml
- Vortex new T9002 culture.
- Put 990ul of the culture into 11 seperate 1.5ml eppendorf tubes to start AHL incubation:
- Label each tube with AHL concentration
- Add AHL from stock
- Record time of AHL inoculation in report sheet.
- Vortex each tube
|
|
we should optimize usage of the 96 plate, more samples needed maybe from different colonies
- Add a 200uL sample from each eppendorf tube to the 96 well plate.
- Do this for 4 repeats following suggested patterning (see above)
- Add 4 x 200uL of growth medium to a well to act as a control.
- Incubate the 96 well palte in a 37oC shaker for 4 hours so GFP expression can reach steady state.
- After the 4 hours add 4 x 200uL of 200x diluted GFP standard solution
<showhide>
- Take 3 readings with a 20 minute interval __HIDER__
<hide>
- IE Readings at 4h, 4h20 and 4h40
</hide> </showhide>
- Taking readings
- Take the plate from shaker and take it to the plate reader room
- Use the Victor3 to measure flourescence and absorbance
<showhide>
- Return the plate to the 37C water bath __HIDER__
<hide>
- There should be a 37oC water bath in the plate reader room with a plastic tub in it. Place the plate in the tub. Don't forget to turn the water bath on before use!
</hide> </showhide> <showhide>
- Save data file from computer. __HIDER__
<hide>
- You'll need a memory stick to save the information to. Unfortunatley the computer isn't networked
</hide> </showhide>
Any questions, see Tom.
Last Updated: Tom 14:53, 14 August 2006 (BST)
Many thanks to Drew Endy and Barry Canton from MIT for providing the protocol on which this is based.
Potential Issues
- We should make a frozen stock of T9002. Maybe 4-5 different colonies from a plate.
- 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?
- 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)
