IGEM:IMPERIAL/2006/Protocols/J37015

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 Shaker
 * Microfuge


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

Protocol (final version)
Culturing overnight (before the testing day):
 * Inoculate all cultures you will be testing using 10 of frozen stock of J37015 (in -80 freezer) by adding 2ml LB containing 50ug/ml Ampicilin.
 * Also inoculate a culture of T9002/J37016 from 10 of stored T9002/J37016 into 2ml LB containing 50/ml Ampicillin.
 * Incubate at 37 for overnight in a shaker.

Following day:
 * Prewarm LB to 37 by placing in the 37 waterbath
 * 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
 * Inoculate fresh 10ml cultures for J37015 and J37019 from the overnight culture to bring back the OD600s to 0.1, use prewarmed LB + Ampicilin.
 * Volume of Overnight Culture X = (0.1/OD600_1)*10ml
 * 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 incubator
 * Remove 1mL of the diluted culture into an eppendorf
 * Spin down the cells and extract supernatant into a new labelled eppendorf
 * Leave the both samples of the supernatant in the fridge for now
 * Incubate new culture (8mL now left) at 37 for 2 hours in a shaker

After the 2 hours in the shaker:
 * Measure and record the OD600_2 of the J37015 and J37019 culture
 * Take 3x 200 samples of the J37015 culture and pipette into a 96 well plate
 * Take 2x 200 samples of the J37019 culture and pipette into a 96 well plate
 * Transfer a 1mL sample of the J37015 from the culture into an eppendorf:
 * Spin down the cells & transfer supernatant into a new labelled eppendorf
 * 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 to the cells
 * Inoculate one of the 25mL J37015 cultures with 5 of 5uM stock concentration of AHL (to get an initial concentration of 1nM AHL)
 * Take all the tubes and the 96 well plate and go over to BCHM

In BCHM lab:
 * Add 4x 200 of 200x diluted GFP control to wells.
 * Add 4x 200 of LB Amp to wells
 * Use the Victor3 to measure GFP output and absorbance
 * Start the clock
 * Place the three 25mL tubes in the 37 shaker
 * Repeat the following steps every 10 minutes for a period of 2hrs (you should end up with a series of about 8-10 measurement time points):
 * Take out the culture of the shaker after the 10min. and make a note of the time
 * Pipette 3x 200µL samples of the J37015 (no AHL added) 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)
 * Use Wallac Victor III to measure OD and fluorescence (Do 2 repeat scans)
 * Always deselect all wells and then only select the row you want to scan
 * Take a 1.5mL sample of the J37015 +AHL and J37015 -AHL culture each
 * Spin down the cells & transfer supernatant into a new labelled eppendorf
 * Place the culture in the shaker for 10 min.

After having taken repeated measurements for 2hrs:
 * Dilute T9002/J37016 to an OD of 0.1 (using formula below) in a new culture of 30ml of a prewarmed LB + Ampicilin.
 * Volume of 2 hour Culture: X = (0.1/OD600_2)*15ml
 * Volume of fresh, prewarmed LB Amp to add: Y = 15 - X  (2 hour Culture)


 * Take 16 x 1ml samples of T9002/J37016 into eppendorfs (or more samples, depending on how many time points were measured + 1 for the morning culture)
 * Spin for 30 seconds
 * Discard the supernatant
 * Resuspend T9002/J37016 cells in 1mL J37015 supernatant of the samples from various time points respectively
 * Take the 1mL sample from the morning culture of J37015 out of the fridge and resuspend T9002/J37026 cells in that supernatant as well (prewarm it if possible)
 * Vortex to resuspend pellet
 * Transfer the different samples into 5mL tubes (label!)
 * Incubate the 5mL tubes in shaker at 37for 4 hours (for GFP to reach steady state)
 * At the same time, prepare J37016 cultures and inoculate with different concentrations of AHL following the J37016 testing protocol
 * Only set up half the volumes given in the table (i.e. a final volume of 1mL)


 * Incubate the 5mL tubes in shaker at 37for 4 hours together with the J37015 samples from above

After 4hrs in the shaker:
 * Take samples out of shaker
 * Pipette the samples into a 96 well plate:
 * Pipette 3x 200&mu;L samples of J37015 (no AHL added) into a 96 well plate
 * Pipette 3x 200&mu;L samples of J37015 +AHL into a 96 well plate
 * Pipette 2x 200&mu;L samples of the J37016 culture into a 96 well plate
 * (Pipette all these sample in one coloumn of the plate)


 * Add pure LB (or LB Amp) and 200 x diluted GFP to 4 wells each as controls
 * 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