IGEM:IMPERIAL/2009/M2/Assays/2.6

Background:

Each of the three testing constructs will result in the production of different colanic acid profiles. Profiles will vary with respect to the amount of colanic acid produced per cell and its protective effect. These two variables must be quantified in order to determine which construct will be most suitable in our final genetic circuit.

Experiment overview:


 * Colanic acid production is measured by packed cell volume (PCV).


 * % of intact cells is measured by fluorescence of supernatent.

Reagents


 * M9 minimal media salts


 * HCL (1M)


 * HSL(3OC6HSL)

Equipment


 * 8 100mL conical flasks flasks


 * 1 1000mL duran

Wet Lab Protocol:

DAY 1:

M9 Minimal Media Preparation:


 * Measure out the following reagents and dissolve them in 1000ml of sterile H20:

Disodium Phosphate = 6.0g

Potassium dihydrogen phosphate = 3.0g

Sodium Chloride = 0.5g

Ammonium Chloride = 1.0g

Carbon source (as determined by Assay 2.2) = 4.0g


 * Split M9 minimal media between 8 conical flasks (100 ml in each).


 * Add a different HSL concentration to each flask (0, 1E-10, 1E-9, 1E-8, 1E-7, 1E-6, 1E-5 and 1E-4 M). This will provide the variable PoPs input. Remember to label flasks with their respective HSL concentrations.


 * Autoclave all media.

DAY 2:

M9 Minimal Media:


 * Dissolve 0.5g of Magnesium Sulphate in 16ml of H20.


 * Using a Minisart® 0.20µm syringe filter, add the 2ml Magnesium Sulphate solution to each of the eight conical flasks containing M9 Minimal Media.

Starter Culture:

- The remaining starter cultures from Assay 2.5 should be used for this Assay.


 * Select a starter culture that corresponds to a sucessfully transformed mucoid colony from Assay 2.5 and pipette 1ml of culture into each of the conical flasks.


 * Incubate the cultures for 24 hours at 37 degrees centigrade on a shaking incubator.


 * After 24 hours, record the OD at 600nm.


 * Add 1ml of culture to each PCV tube and spin at 5000 rpm for 1 minute.


 * If the PCV is greater than 5 micro litres, load less cell suspension and spin again.


 * Use HCl to acidify each culture (to pH 2) to simulate gastric conditions; then incubate for 2 hours (37 degrees centigrade).


 * At 20 minute intervlas, take OD (600nm). Additionally take 1ml samples, transfer to an eppindorf and place on ice.


 * After the 2 hour incubation period, transfer the samples to a 96 well plate and measure fluorescence of sample. A drop in fluoresence is indicative of denatured GFP.