IGEM:IMPERIAL/2006/Protocols/J37015

Part J37015: Prey Cell Positive Feedback Characterisation

Motivation

The motivation behind this test construct is to assess the length and amplitude of the exponential phase. This is essential to comply with the Lokta-Volterra constraints.

We need to characterise the positive feedback loop to work out the intrinsic growth rate of the prey JohnChattaway 05:19, 24 August 2006 (EDT)

Equipment & Materials

• Equipment
  • Wallac Victor 3 Multi-Well Fluorimeter
  • Eppendorf Tubes
  • Gilson Pippettes
  • 37[[:Category:{{{1}}}|{{{1}}}]] Shaker
  • Microfuge

• Materials
  • E.coli Growth Medium - LB w/Ampicillin
  • E.coli Culture Containing T9002
  • E.coli Culture Containing J37015
  • GFP Standard Solution

Protocol

Culturing overnight (before the testing day): <showhide>

• Inoculate all cultures you will be testing using 10μL of frozen stock of J37015 (in -80[[:Category:{{{1}}}|{{{1}}}]] freezer) by adding 2ml LB containing 50ug/ml Ampicilin. __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

</hide> </showhide> <showhide>

• Also inoculate a culture of T9002/J37016 from 10μL of stored T9002/J37016 into 2ml LB containing 50μg/ml Ampicillin. __HIDER__

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The T9002/J37016 cells will be used for the AHL assay

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• Incubate at 37[[:Category:{{{1}}}|{{{1}}}]] for overnight in a shaker.

Following day: <showhide>

• Prewarm LB to 37[[:Category:{{{1}}}|{{{1}}}]] by placing in the 37[[:Category:{{{1}}}|{{{1}}}]] waterbath __HIDER__

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This is done now so that when you need to use the media later it'll be prewarmed

</hide></showhide>

• Measure and record the OD600 for all cultures.
• Take a 1mL sample of the J37015 culture
• Spin down the cells & extract supernatant into a labelled eppendorf

<showhide>

• Inoculate fresh 10ml cultures for J37015 and J37019 from the overnight culture to bring back the OD600s to 0.1, use prewarmed LB + Ampicilin. __HIDER__

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Using prewarmed LB prevents a temperature shock to the culture, which would increase lag time

</hide></showhide>

• Volume of Overnight Culture X = (0.1/OD600_1)*25ml
• Volume of LB Amp to add Y = 10mL - X (Overnight Culture)

• Also dilute the overnight culture of T9002/J37016 into 16mL to an OD 0.1
• Put this new culture back into the shaker for 2hrs
• Return LB Amp to 37[[:Category:{{{1}}}|{{{1}}}]] incubator
• Remove 1mL of the diluted culture into an eppendorf
• Spin down the cells and extract supernatant into a new labelled eppendorf

<showhide>

• Leave the both samples of the supernatant in the fridge for now __HIDER__

<hide>

The supernatant is to be tested for AHL concentration later (using the J37016 assay). This is to test whether there has been any leaky expression of AHL from overnight. Since half life of AHL is relatively slow, any AHL produced overnight should still be present the next day.

</hide></showhide> <showhide>

• Incubate new culture (8mL now left) at 37[[:Category:{{{1}}}|{{{1}}}]] for 2 hours in a shaker __HIDER__

<hide>

This returns cells to exponential phase from stationary phase

</hide></showhide>
After the 2 hours in the shaker:

• Measure and record the OD600_2 of the J37015 and J37019 culture
• Take 4x 200μL samples of the J37015 culture and pipette into a 96 well plate
• Transfer a 1mL sample of the J37015 from the culture into an eppendorf:

<showhide>

• Spin down the cells & transfer supernatant into a new labelled eppendorf__HIDER__

<hide>

To get inital AHL concentration

</hide></showhide>

• Add 25mL fresh, prewarmed LB Amp to the cells
• Dilute fresh 2 x 25ml cultures for J37015 and dilute a 25mL cuture of J37019 with LB Amp to bring back the OD600s to 0.1, use prewarmed LB + Ampicilin:
  • Pipette the volume of J37015/J37019 needed such that it will be diluted to OD 0.1 in 25mL into a 50mL Falcon tube
  • Spin down the cells & discard supernatant
  • Add 25mL prewarmed LB Amp

<showhide>

• Inoculate one of the 25mL J37015 cultures with 5μL of 5uM stock concentration of AHL (to get an initial concentration of 1nM AHL)__HIDER__

<hide>

This is done to make sure that AHL levels start off in the detectable range of the J37016 assay.

</hide></showhide>

• Take all the tubes and the 96 well plate and go over to BCHM

In BCHM lab:

• Add 4x 200μL of 200x diluted GFP control to wells.
• Add 4x 200μL of LB Amp to wells

<showhide>

• Use the Victor3 to measure GFP output and absorbance __HIDER__

<hide>

This is to assess the state of the positive feedback loop which should be unactivated ???

</hide> </showhide>

• Start the clock
• Place the three 25mL tubes in the 37[[:Category:{{{1}}}|{{{1}}}]] shaker
• Repeat the following steps every 10 minutes for a period of 1h (or 2hrs ?):
  • Place the culture in the shaker for 10 min.
  • Pipette 3x 200μL samples of the J37015 +AHL culture into a 96 well plate
  • Pipette 3x 200μL samples of the J37015 -AHL culture into a 96 well plate
  • Pipette 2x 200μL samples of the J37019 culture into a 96 well plate

(Pipette all these sample in one coloumn of the plate)

<showhide>

• Dilute T9002/J37016 to an OD of 0.1 (using formula below) in a new culture of 15ml of a prewarmed LB + Ampicilin. __HIDER__

<hide>

This dilution gives a standard OD to which to inoculate the culture with AHL (in this case 0.1). Inoculating at different ODs is known to give different results, so it is important a standard OD is used

</hide></showhide>

• Volume of 2 hour Culture: X = (0.1/OD600_2)*15ml
• Volume of fresh LB Amp to add: Y = 15 - X (2 hour Culture)

• Take 1ml samples of T9002 into eppendorfs
• Spin for 20 seconds
• Discard the supernatant
• Resuspend T9002 cells in 1mL J37015 supernatant
• Take the 2mL sample from the morning culture of J37015 out of the fridge and resuspend T9002/J37026 in that supernatant as well (prewarm it if possible)
• Vortex to resuspend pellet
• Incubate plate in shaker at 37[[:Category:{{{1}}}|{{{1}}}]]for 4 hours (for GFP to reach steady state)

After 4hrs in the shaker:

• Pippete 4x 200 μL samples of the resuspended T9002/J37016 cells into a 96 well plate
• Measure and record OD and flourescence using Wallac Victor III in BCHM

This should give you a series of GFP flourescence readings for each time point and each culture

Potential Issues

• Cells have to spun down, which makes them a little unhappy
• Unsure of what level of repression we'll get from the various control mechanisms
• Unsure of the speed of the positive feedback loop

comments from Vincent

• Depending on the strengh of the positive feedback, it might be interesting to explore different dilutions to begin with.

To do list

Assess LuxI through out the experiment