Table 1.
Composition of regular M9 and the new M9 ++ medium for expression of U-[15N/2H]/Ileδ1-[13CH3];Leu/Val-[13CH3] labeled proteins
| Regular M9 medium (1 L) | M9 ++ medium (100 ml) | |
|---|---|---|
| K2HPO4 | 10.0 g | 1.9 g |
| KH2PO4 | 13.0 g | 0.5 g |
| Na2HPO4 | 9.0 g | 0.9 g |
| K2SO4 | 2.4 g | 0.24 g |
| D-d7-Glucosea | 2.5 g | 1.8 g |
| NH4Cla | 1.0 g | 0.5 g |
| Trace element solutionb | 10.0 ml | 100 μl |
| MEMc | 10.0 ml | – |
| 1 M MgCl4 | 10.0 ml | 100 μl |
| LB/D2O | – | 0.1 ml |
| α-ketoisovaleric acidd | 100 mg | 32 mg |
| α-ketobutyric acidd | 50 mg | 16 mg |
For perdeuteration d7-D-glucose is the sole carbon source; for perdeuteration and uniform 13C-labeling 13C6/d7-D-glucose would be used instead.15NH4Cl is used as the sole nitrogen source
The trace element solution is that described by (Cai et al. 1998) and comprises the following per 100 ml: 0.6 g FeSO4 (7H2O), 0.6 g CaCl2 (2H2O), 0.12 g MnCl2 (4H2O), 0.08 g CoCl2 (6H2O), 0.07 g ZnSO4 (7H2O), 0.03 g CuCl2 (2H2O), 2 mg H3BO4, 0.025 g (NH4)6Mo7O24 (4H2O), 0.5 g ethylenediaminetetraacetic acid (EDTA)
MEM vitamins 100 × solution from Corning (VWR catalog number 45000–702) was lyophilized and dissolved back in the same volume of D2O
α-ketoisovaleric acid (1,2,3,4-13C4, 98 atom %; 3,4′,4′,4′-d4, 98 atom %) and α-ketobutyric (13C4, 98%; 3,3-d2, 98 atom %) are only added 1 h prior to induction