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

Use the diluted culture!!!!

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• Inoculate all cultures you will be testing using diluted stocks from 10μL of J37015 in 2ml LB/M9 growth medium 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

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• Also inoculate a culture of T9002 from 10μL of stored T9002 into 2ml LB/M9 growth medium containing 50μg/ml Ampicillin. __HIDER__

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The T9002 cells will be used for the AHL assay

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

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• 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 on it'll be prewarmed

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• Measure and record the OD600 for both cultures.

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• Inoculate fresh 10ml cultures for both J37015 and T9002 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

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

• Return LB Amp to 37[[:Category:{{{1}}}|{{{1}}}]] incubator
• Remove 2x 1mL of the diluted culture into 2 eppendorfs
• Spin down the cells and extract supernatant into a small white 5mL tube (label!)

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• Leave the supernatant in the fridge for now __HIDER__

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The supernatant is to be tested for fluorescence later. This is to test whether there has been any leaky expression of AHL (and thus GFP) from overnight.

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

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This returns cells to exponential phase from stationary phase

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After the 2 hours in the shaker:

• Measure and record the OD600_2 of the J37015 culture
• Transfer a 1mL sample from the culture into an eppendorf
• Spin down the cells
• Take 4x 200μL samples from the supernatant of the J37015 culture and pipette into a 96 well plate
• Take the 2mL sample of the fridge
• Add 4x 200μL of 200x diluted GFP control to wells.
• Add 4x 200μL of LB Amp to wells

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• Use the Victor3 to measure GFP output and absorbance __HIDER__

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

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• Having returned to the teaching lab, take 1ml of J37015 cell culture into a cuvette and measure OD600
• Empty the curvette into a labelled eppendorf
• Spin down cells in the eppendorf for 20 seconds
• Pipette supernatant from J37015 into newly labelled eppendorf tube To get inital AHL conc?
• (To the J37015 cultures add Theophylline for a final concentration of 10mM for J37015RS or 1mM of IPTG for J37015Cre add 70ul of 1M theophyline) __HIDER__

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This allows us to activate the feedback loop

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• Start the clock

• Take 1ml of J37015 cell culture into a cuvette and measure OD600
• Empty the cuvette into a labelled eppendorf
• Spin down cells in the eppendorf for 20 seconds
• Pipette supernatant from J37015 into newly labelled eppendorf tube
• Repeat these steps to take samples every 10 minutes
• After a period of 60 minutes, stop taking samples

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• Dilute T9002 for an OD of 0.1 (using formula below) in a new culture of 15ml of a prewarmed LB/M9 + Ampicilin. __HIDER__

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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

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• 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 J37015 supernatant
• Vortex to resuspend pellet
• Pippete 4x 200 μL samples of the resuspended T9002 cells into a 96 well plate
• Incubate plate in shaker at 37[[:Category:{{{1}}}|{{{1}}}]]
• Wait for 4 hours for GFP to reach steady state
• Measure and record OD and flourescence

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