Kevin Gardner - September 1998

notes:
1.  This is one of the simpler M9 recipes in circulation, one that I picked up while working as a postdoc in Toronto.  It has worked well in our hands for expressing a variety of proteins from several strains of E. coli, most usually BL21(DE3).  To overexpress proteins containing certain cofactors, including metal ions, consider supplementing this growth media with those cofactors or their precursors.

2.  Deuteration: this protocol can be readily adapted for use with D2O for the production of deuterated proteins.  For general information about this, please check here (Gardner and Kay, 1998) or contact KG at Kevin.Gardner@utsouthwestern.edu.

sources of (stable) isotopically-labelled compounds: 
for the protocol listed below, 15N ammonium chloride and 13C glucose are used.  These can be purchased from a number of suppliers, including (listed in alphabetical order):

Cambridge Isotope Labs (purchased Spectra Stable Isotopes - July 2008)
Sigma-Aldrich (formerly Isotec)
Silantes

protocol:
1).  prepare stock solutions, dissolving each group of components into the specified amount of water and autoclaving:

10x M9 salts (dissolve in 1L H2O, adjust pH to 7.4)
67.8g Na2HPO4 (anhydrous)
30g KH2PO4
5g NaCl

10x ammonium chloride (dissolve in 1L H2O)
10g NH4Cl [natural abundance 15N]

glucose stock (dissolve in 100mL H2O)
20g glucose (Cf: 20%) [natural abundance 13C]

CaCl2 * 2H2O (dissolve in 100mL H2O)
1.11g CaCl2 (Cf: 0.1M)

MgSO4*7 H2O (dissolve in 100mL H2O)
24.6g MgSO4 (Cf: 1.0M)

H2O (approx. 1.2L)

2).  prepare additional stock solutions at the specified concentrations and sterile-filter through a 0.22um filter:
thiamine (10mg/mL)

biotin (10mg/mL) --- as this is above the solubility limit of biotin, do not sterile filter this solution.  Simply make solution up with previously sterilized water.

ampicillin or other antibiotic for plasmid selection

3).  from a freshly/recently-transformed plate of E. coli containing plasmid for overexpression of desired protein, select a single colony and use it to innoculate a 3mL culture of LB with appropriate antibiotic.  Grow for ca. 3-5hr at 37C with vigorous shaking, until culture is visibly turbid.  (alternatively: grow LB culture overnight, preferably at 25-30C to avoid culture becoming overgrown for an extended period of time)

4).  prepare 50mL of M9 media as follows, in a 250mL flask:

5mL 10x M9 salts
5mL 10x ammonium chloride (natural abundance)
750uL 20% glucose (natural abundance)
50uL of each CaCl2, MgSO4, thiamine and biotin
antibiotic at working concentration
autoclaved H2O to 50mL
Prewarm media to 37C.  While warming, centrifuge LB culture (5', 4000xg, 30C) and discard supernatant. 

5).   Resuspend cell pellet in 50mL M9 media, for a starting OD(600nm) of approximately 0.03-0.1.  Let culture grow for approximately 3-4hr at 37C until the culture is mid-log, OD(600nm)~0.5-0.8.  Typical doubling times for BL21(DE3) in this media is approximately 60-75 minutes.

6).  prepare 1L of M9 media as follows, in a 2.8L Fernbach flask:

 
100mL 10x M9 salts
100mL 10x ammonium chloride OR  1g 15NH4Cl (dissolved in 10mL H2O and sterile filtered)
15mL 20% glucose OR 3g 13C glucose (dissolved in 10mL H2O and sterile filtered)
1mL of each CaCl2, MgSO4, thiamine and biotin
antibiotic at working concentration
autoclaved H2O to 1L
(n.b.  significantly improved yields can be obtained by spiking this media with complex rich media, often at lower levels than are suggested by the manufacturers.  We have had good success with CIL's 10x 15N/13C Bioexpress media, using only 10mL Bioexpress/L of M9 instead of the full-strength 100mL/L (Holdeman and Gardner, J. Bio. NMR 21(2001): 383).

Prewarm media to 37C.  While warming, centrifuge M9 culture (5', 4000xg, 30C) and discard supernatant.

7).  Resuspend cell pellet in 1L M9 media, for a starting OD(600nm) of approximately 0.03-0.08.  Let culture grow for approximately 3-4hr at 37C until the culture is mid-log, OD(600nm)~0.5-0.8.  Typical doubling times for BL21(DE3) in this media is approximately 60-75 minutes.  Induce protein expression as desired.  Using our typical expression protocol (0.5mM IPTG, overnight at 20C), typical final OD(600nm) range from 2.0-2.5.