Protocol for 13C and 15N-Isotope Labeling in Minimal Media Be sure to use freshly transformed colonies.

Start 5 mL culture in LB in the morning. Inoculate 50 mL of minimal media in 250 mL flask with 1 mL of 5 mL starter culture and let grow overnight. In the morning, inoculate 4 X 250 mL in 2 L flasks of minimal media with 4 X 12.5 mL of overnight culture. Induce when cultures have an OD600nm of 0.5 and 0.6 and harvest the cells when the OD600nm is between 1.1 and 1.2 or when they stop growing (whichever comes first). M9 Medium For 1L, 4 X ~217 mL in 2L flasks: - dH2O - M9 salts 10X 867 100 mL mL

Autoclave and add the following (filter-sterilize all solutions): - 1 M MgSO4 - Vitamin Mix * (SIGMA # B6891) - Solution Q - Antibiotic - Glucose or 13C Glucose - NH4Cl or 15N NH4Cl 2 10 2 1 4 1 mL mL mL mL g g

* Vitamin Mix can also be made using the attached recipe. Solutions M9 salts 10X (for 1L) - Na2HPO4 - KH2PO4 - NaCl Adjust pH to 7.1 7.3 67.9 30 5 g g g

Solution Q (for 1L add in the following order) - 5M HCl 8 mL (Be sure to dilute in H2O before adding metals - FeCl2.4H2O 5 g . - CaCl2 2H2O 184 mg - H3BO3 64 mg . - CoCl2 6H2O 18 mg - CuCl2.2H2O 4 mg - ZnCl2 340 mg

- Na2MoO4.2H2O - MnCl2.2H2O

605 40

mg mg

Vitamin Mix (sterile filter and store @ -20C) (for 100 mL) - Thiamine 50 mg - d-Biotin 10 mg - Choline chloride 10 mg - Folic acid 10 mg - Nicotinamide10 mg - D-pantothenic acid 10 mg calcium salt - Pyridoxine HCl 10 mg - Riboflavin 1 mg