Team:Cambridge/Protocols/MgFreeCells

Production of Magnesium Free Cells

Risk Assessment

If you wish to test a component that is sensitive to magnesium, you will need to ensure that your cells do not contain any magnesium beforehand. If your cells do contain magnesium before you begin any characterization, your results will be skewed.

A series of growth curves for both e.coli and bacillus can be found in our lab-book, demonstrating the different magnesium requirements of these two species. In particular, Bacillus will require a considerable concentration of magnesium (over 20 μM) before it will grow well, whereas e.coli appears capable of scavenging more from its environment and previous generations. E.coli will therefore grow more successfully under conditions of extremely low magnesium, and is likely to give better readings for your component at these levels.

We have had successes with two approaches:

Before you start

• Make up medium B, however be careful to remove all traces of magnesium from the mix (you will need to remove the MgSO4.7H2O from the bacillus salts and add no MgCl2 in the final step). This will be your defined medium, lacking any magnesium ions.
• Medium B autofluoresces, so if you are testing a part that will alter expression of GFP, you will need to use M9 medium instead.

Approach 1

• Step 1: Grow your cells to a high (0.6 or greater) OD620 in normal LB overnight.
• Step 2: Take 2ml of this culture and centrifuge to make a pellet. Remove supernatant with a pipette.
• Step 3: Resuspend pellet in 2ml of medium. Incubate for 20 mins to allow magnesium to diffuse out of cells into medium.
• Step 4: Centrifuge to make a pellet and remove supernatant. Resuspend pellet in 2ml medium.
• Step 5: Your cells are now ready to be tested. If preparing a plate reader assay, dilute 200 times into new medium and allow to grow for 2 hours at 37 °C before adding magnesium.
• This technique will produce a magnesium free culture with a relatively high cell density. However, traces of magnesium may still remain within the cells from earlier generations. This approach is particularly useful if you have a culture of cells in LB which you need to test right away.

Approach 2

• Step 1: Grow your cells in medium B overnight. The resultant culture will not be very turbid, because magnesium is required for cellular proliferation.
• Step 2: Dilute cell culture 200 times in new medium B. Cells are now ready to be used for assays.
• While this technique will produce cells containing almost no magnesium, the low cell density produced may be an issue for some assays. The growth curves at different magnesium concentrations were made with approach 1, so you may need to repeat such experiments with this second approach to get a reasonable comparison.