I am trying to screen a particular Zinc sensor that I am developing. I want to express the variants in E.coli and quickly record the fluorescence with added metal after lysing the cells to check for the sensor's efficacy.
Long short story, I am looking for the ideal media to grow the cells in. Additionally, I am wondering if the addition of a generic chelator after lysing the cells would be better in this case?
As your question suggests, in order to grow or achieve a relatively Zinc metal free coli culture, you need to grow it in M9 media.
Make M9 salts
To make M9 Salts aliquot 800ml H2O and add
64g Na2HPO4-7H2O
15g KH2PO4
2.5g NaCl
5.0g NH4Cl
Stir until dissolved
Adjust to 1000ml with distilled H2O
Sterilize by autoclaving
Measure ~700ml of distilled H2O (sterile)
Add 200ml of M9 salts
Add 2ml of 1M MgSO4 (sterile)
Add 20 ml of 20% glucose (or other carbon source)
Add 100ul of 1M CaCl2 (sterile)
Optionally, you may add heavy ,metal solution. But in this case, since you don;t need many metals. The above solution is ideal!
Adjust to 1000ml with distilled H2O
In addition, if you want to be really sure of no zinc, then I would suggest an Atomic absorption spectroscopy to go along with the above method (optional).
In addition, I would Chelex-filter the medium as there are always slight contaminations of ions in the salts.
I doubt you will be able to get a true zinc-free medium where E. coli can grow (it is an essential cofactor for many enzymes). However, if the zinc sensors you are developing will not be thrown off by traces of zinc at the ppm level, then any chemically defined medium prepared without zinc salts, using type I or II reagent grade water with high quality reagents in acid-washed glassware -or simply disposable plasticware- will do the trick. M9 medium as suggested by Anirudh Shanbhag above is a good starting point.
I would not, however, treat the medium with Chelex, as it will deplete it of many other necessary ions.
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