Team:Imperial College London/Wetlab/Protocols/ColanicAcid

Phase 1, Growing up your cells:

In our system, colanic acid is only produced during the utilization of a secondary carbon source. For this reason we must use minimal media for this assay such that we are able to define the carbon source used.

DAY 1:

AM:

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

Secondary carbon source (as determined by the Top Ten cell growth assay) = 4.0g

Casamino acids =?

Leave out for autoclaving (before 12:30)

PM:

Collect media from autoclave (at 5pm)

M9 Minimal Media Innoculation:

• Add 4.0g of filter the sterilised secondary carbon source as well as 0.5g magnesium sulphate.

• Spit the media between two 500ml autoclaved flasks.

• Innoculate one of the flasks of M9 Minimal Media with 7ml of Top Ten starter culture carrying the encapsulation construct. Innoculate the second flask of M9 Minimal Media with 7ml of Top Ten starter culture not transformed with the encapsulation construct to serve as a control. Incubate both flasks at ninteen degrees centigrade in a shaking incubator.

Phase 2, Quantifying Colanic Acid Production:

In brief, colanic acid production can be quantified from two pieces of information: the packed cell volume and the OD of a culture.

DAY 2:

AM:

• After 24 hours of incubation, pippette 1ml of culture from each of the two flasks into PCV tubes.

• Spin the PCV tube at 13,000 rpm for 1 minute and record the volume of the pellet.

• Take another 1ml sample from each culture and measure the OD at 600nm.

• Divide the PCVs by the OD and subtract the value obtained for the controls from the value obtained from the transformed cells. This relates to the average volume of colanic acid produced per cell.

• From a stock solution of concentrated HCl of pH 0.5 prepare the following 4ml dillutions by adding ddH20 where appropriate.

pH = ddH20

pH = 1.0

pH = 1.5

pH = 2.0

pH = 2.5

pH = 3.0

pH = 3.5

pH = 4.5

• Each of these eight dilutions should correspond to a different row on the plate as shown below:

PM:

• Spin down 40mls of solution from each flask for ten minutes at 4 degrees centigrade.

• Pour off the supernatent and resuspend each of the pellets in 5 mls of ddH20.

• Transfer 100ul from the resuspended pellet of transformed cells into the first 6 columns and 100ul from the resuspended pellet of untransformed control cells into the second 6 columns of a ninety six well plate. See the above image for clarification.

• Using a parallel pippette, transfer 100ul of acid solution to each well.

• Place the plate in the plate reader and collect data for a six hour period. Data of interest should be OD and GFP fluorescence.

• Since colanic acid production takes a long time, the Day 2 protocol should be repeated for a total of three days to determine the optimum colanic acid profile. Note, this requres that the whole experiment be started on a Monday.