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:

M9 Minimal Salts = 11.28g

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

Casamino acids = 2g

Leave out for autoclaving (before 12:30)

PM:

Collect media from autoclave (at 5pm)

M9 Minimal Media Innoculation:

• Add 4.0g of the filter sterilised secondary carbon source as well as 0.5g of filter sterilised magnesium sulphate. Filtering should be done using a Minisart® 0.20µm syringe filter.

• 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 28°C 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 gives the average volume of colanic acid produced per cell.

Phase 3, Quantifying the protective effect of colanic acid:

This stage of the assay works on the principle that acid will lyse cells resulting the the denaturation of internal GFP marked by a fall in fluorescence over time. The better the protective effect of the colanic acid coat, the longer the fluorescence will last.

• From a stock solution of concentrated HCl of pH 0.5 prepare the following 4ml dillutions by adding ddH20. This should be done using a pH meter. These dilutions should be prepared in 15ml Falcon tubes. Note, Conc HCl is dangerous and should be treated with great care.

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 correspond to a different row on the ninety six well plate as shown below:

PM:

• Using a centrifuge, spin down 40mls of solution from each flask for ten minutes at 4 degrees centigrade and sixty thousand rpm.

• 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. This step should be done as quickly as possible. To facilitate this, it is advised that the acid solutions are first pippetted into eppindorfs.

• 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.