IGEM:IMPERIAL/2006/Protocols/T9002
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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
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- 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__
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- 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
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- Prewarm 16ml of LB/M9 to 37oC by placing in the 37oC incubator overnight __HIDER__
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- 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
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- Following day, measure and record OD600_1 in report sheet
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- Inoculate a 16ml fresh culture from the o/n to bring back the OD600 to 0.1, use the prewarmed LB/M9 + Ampicilin in incubator. __HIDER__
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- Using prewarmed LB/M9 prevents a temperature shock to the culture, which would increase lag time
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- Volume of original culture used to inoculate new culture = (0.1/OD600_1)*16ml
- Volume of prewarmed medium to use = 16 - Result from above
- Return LB/M9 to 37oC incubator
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- Incubate new culture at 37oC for 2 hours in a shaker __HIDER__
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- This returns cells to exponential phase from stationary phase
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- Prewarm 30ml of LB/M9 in the 30oC incubator
- After the 2 hours measure and record the OD600_2 in report sheet
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- Dilute again for an OD of 0.1 in a new culture of 30ml of a prewarmed LB/M9 + Ampicilin. __HIDER__
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- 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
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- Volume of original culture used to inoculate new culture = (0.1/OD600_1)*30ml
- Volume of prewarmed medium to use = 30 - Result from above
- Vortex new T9002 culture.
- Put 20ul of the AHL into 11 seperate 5ml white capped tubes
- To start AHL incubation:
- Label each tube with AHL concentration
- Add T9002 samples
- Record time of inoculation in report sheet.
- Vortex each tube
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- Add a 200uL sample from each eppendorf tube to the 96 well plate.
- Do this for 8 repeats following suggested patterning (see above)
- Add 4 x 200uL of growth medium to a well to act as a control.
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- Incubate the 96 well palte in a 37oC shaker set to 100rpm for 4 hours so GFP expression can reach steady state. __HIDER__
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- Tape the 96 well plate to an upturned plastic tub. You can then put the plastic tub in the shaker, going over the prongs
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- After the 4 hours take the plate and an ependorf to BCHEM
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- Add 190ul of ultra pure water to the ependorf, together with 10ul of undiluted GFP standard solution __HIDER__
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- The undiluted GFP is in the BCHM Level 6 Cold Room on our shelf. It is in a small grey plastic box. Pippettes, tips and ultrapure water are located on a shelf above the electroporation machine in the plate reader room.
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- Add 4 x 200uL of the 200x diluted GFP standard solution to the wells following the suggested patterning
- Take a reading
- Take the plate to the plate reader room
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- Use the Victor3 to measure flourescence and absorbance __HIDER__
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- Use the preprogrammed Assay under the 'Students' folder called GFP + Abs490
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- Repeat the measurment a further two times straight after each other __HIDER__
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- This to assess the variability of the machine
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- Save data file from computer. __HIDER__
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- You'll need a memory stick to save the information to. Unfortunatley the computer isn't networked
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- Copy and paste the data into a T9002 Data Spreadsheet
Any questions, see Tom.
Last Updated: Tom 15:06, 18 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)
