IGEM:IMPERIAL/2009/M2/Assays/2.2

Background:
Once the glucose in the media has been consumed, the chassis will utilise a secondary carbon source to faciliate the production of colanic acid. Assay 2.2 serves to determine the secondary carbon source that is most suited to colanic acid production.

Equipment Required:

 * Minisart® 0.20µm syringe filter
 * Incubator
 * Conical flask. Volume = at least 100ml (x7)
 * Duran. Volume = 1000ml (x1)

Reagents Required:
Carbon Sources:
 * Glucose
 * Galactose
 * Maltose
 * Arabinose
 * Xylose
 * Rhamnose
 * Lactose

Chemical Induction of Colanic Acid:
 * FPA (14.65 mg)

M9 Minimal Media:
 * Disodium Phosphate (Na2HPO4) (6.0g)
 * Potassium dihydrogen phosphate (KH2PO4) (3.0g)
 * Sodium Chloride (NaCl) (0.5g)
 * Ammonium Chloride (NH4Cl) (1.0g)
 * Magnesium Sulphate (MgSO4) (0.35g)

CALCULATIONS:
According to the literature, liquid media should contain 8 X 10-5 M FPA for the induction of colanic acid biosynthesis.

According to the protocol, we require a 1000ml M9 stock solution to make the culture media.

Therefore this stock solution should contain 8 X 10-5 moles of FPA.

Since the FPA molar mass is 183.18 grams per mole, we require 14.65 mg of FPA for the protocol.

Preparation of M9 Media:
Minimal M9 media is a defined minimal growth media suited to optical density based studies on account of its low absorbance and autofluoresence.

MgSO4 solution precipitates when heated and therefore cannot be autoclaved. To maintain sterility, MgSO4 solution should be filter sterilised (Minisart® 0.20µm syringe filter) and added after the other chemicals had been dissolved, mixed and autoclaved. To support bacterial growth, M9 medium requires the addition of a carbon source.


 * 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


 * Label 8 conical flasks according to carbon source as shown below:

1) Glucose

2) Galactose

3) Maltose

4) Arabinose

5) Xylose

6) Rhamnose

7) Lactose

8) No-carbon source

M9 Minimal Media Preparation:
 * Decant 100ml of M9 minimal media into each of the 8 conical flasks
 * Measure out and add 0.4 grams of each carbon source and add it to the conical flask with the corresponding label.
 * Autoclave the M9 media to ensure sterility.
 * Dissolve 0.4g of Magnesium Sulphate in 16ml of H20.
 * Using a Minisart® 0.20µm syringe filter, add 2ml of Magnesium Sulphate solution to each conical flask.

Media Innoculation:

 * Innoculate each M9 Minimal media with 2.5ml of appropriate starter culture. Note, sufficient starter culture culture should be left over from Assay 2.1.


 * Take the initial OD (@600nm) and then place in a shaking incubator at 37 degrees centigrade.


 * After 24 hours, add 1ml of each culture to a PCV tube and spin at 5000 rpm for 1 minute. Record the culture volume added to each PCV tube and the corresponding PCV in microlitres following centrifugation.


 * If the PCV is greater than 5 micro litres, load less cell suspension and spin again (Be careful to note the new volume added if this is the case). If the PCV is less than 5 micro litres, record the volume.


 * Also measure the OD of the culture at 600nm to determine the final population density.

Data Treatment:
For each carbon source, this assay will produce two pieces of information:

1) PCV

2) OD (@600nm)

By dividing the PCV by the OD, we will obtain a relative measure of the amount of colanic acid produced per cell. The secondary carbon source that provides the greatest PCV/OD ratio will be selected for use in our Induction Media.

Provided that there is not a massive decline in colanic acid production in response to utilisation of a non-glucose carbohydrate, then we are justified in using a CRP depenent promoter for the induction of M2. If however we encounter a failure to produce colanic acid in the absence of glucose, then we will have to re-think the induction of M2. There is no evidence in the literature for the latter senario but I think that it is good to be safe and investigate.