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Methods:GGM (glycerol-glycerophosphate medium)

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Contents

Purpose

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"GGM is buffered with MES, which has minimal metal chelating properties, and uses organic phosphate as the phosphate source to minimize formation of insoluble metal phosphates."[1]

Materials Needed

Protocol

The final concentrations are: MES (40.0 mm), NH4Cl (18.7 mm), KCl (13.4 mm), β-glycerophosphate (7.64 mm), glycerol (5.00 mm), K2SO4 (4.99 mm), MgCl2 (1.00 mm), EDTA (134 μm), CaCl2·2H2O (68.0 μm), FeCl3·6H2O (18.5 μm), ZnO (6.14 μm), H3BO3 (1.62 μm), CuCl2·2H2O (587 nm), Co(NO3)2·6H2O (344 nm), and (NH4)6Mo7O24·4H2O (80.9 nm) in MilliQ water (Millipore). Bulk elements (MES, NH4Cl, KCl, K2SO4, and glycerol in MilliQ water at pH 7.4 (batch growth) or 7.6 (continuous culture)) were passed through a column containing Chelex-100 ion exchange resin (Bio-Rad) to remove contaminating cations. Trace elements (with or without Zn2+ as necessary) and a CaCl2 solution were then added to give the final concentrations shown above prior to autoclaving. After autoclaving, MgCl2 and β-glycerophosphate were added at the final concentrations shown. All of the chemicals were of AnalaR grade purity or higher. Chelex-100 was packed into a Bio-Rad Glass Econo-column (~120 × 25 mm) that had previously been soaked in 3.5% nitric acid for 5 days.[1] [2]

Usage Examples

Zinc depletion of E. coli [1]

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References

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  1. 1.0 1.1 1.2 Graham, AI et al. (2009) Severe zinc depletion of Escherichia coli: roles for high affinity zinc binding by ZinT, zinc transport and zinc-independent proteins. J. Biol. Chem. 284 18377-89 PubMed EcoliWiki page
  2. Beard, SJ et al. (1997) Zinc(II) tolerance in Escherichia coli K-12: evidence that the zntA gene (o732) encodes a cation transport ATPase. Mol. Microbiol. 25 883-91 PubMed EcoliWiki page