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  • Recommended media and expression conditions for T7 Express Crystal (C3022)

    Protocol

    1. General notes: T7 Express Crystal (C3022) is a metB1 derivative of T7 Express (C2566). 50 ug/mL L-seleno-methionine may be substituted with 100 ug/mL of D/L isomer mix. Sigma product (S-3875) is recommended-prepare a 10 mg/mL stock in dH20. For enhanced growth, add 0.0002% ferric ammonium citrate to minimal media. Fisher Scientific product #I72-500 is recommended as this product also provides trace metals-Filter sterilize ferric ammonium citrate. Transformed cells may be plated on rich agar. Inoculate a single colony into 10 mL minimal media plus 50 ug/mL L-methionine – grow for up to 24 hrs to achieve a saturated culture. Inoculate starter culture at a 1:100 dilution into expression media. 

    2. Maximum Seleno-methionine incorporation (regulated expression systems): Use an expression vector with a T7-lac promoter or other regulated system. Grow cells in minimal media (plus 50 ug/mL L-methionine) until mid-log phase (OD=0.6-0.8). Then pellet cells and resuspend in fresh, pre-warmed minimal media without Met or SeMet. Grow for 2.5 hours at 37°C to deplete cells of methionine (longer if using a lower temperature). Add 50 ug/mL L-seleno-methionine (or 100 ug/mL of D/L isomer mix). Incubate for 30 minutes and then add inducer. Induce for 3 hours to overnight. 

    3. Seleno-methionine labeling using constitutive expression systems: Prepare minimal media with a 10:50 ratio of L-Met to L-SeMet to achieve a maximal growth rate. For example, add 10 ug/mL L-methionine and 100 ug/mL D/L-selenomethionine. This ratio will result in approximately 83% SeMet labeling of expressed protein. Use the same expression conditions previously optimized for your protein of interest. If a higher level of SeMet labeling is desired, spike with 100 ug/mL D/L-selenomethionine at mid-log to late-log phase of growth. 

    4. Minimal media for T7 Express Crystal (per Liter): 
      200 mL of 5X M9 salts (Sambrook et al.) 
      20 mL 20% glucose (0.4% final conc.) 
      1 mL of 1M MgSO4 (1 mM final conc.) 
      0.1 mL of 1M CaCl2 (0.1 mM final conc.) 
      0.2 mL of 1% ferric ammonium citrate (0.0002% final conc.) 
      Met, SeMet or Met/SeMet mix (see above) 
      Antibiotic to select plasmid(s) Sterile 
      dH20 to 1 Liter 


      Reference: 
      Lambert AR et al. (2008) Structures of the Rare-cutting Restriction Endonuclease NotI Reveal a Unique Metal Binding Fold Involved in DNA Binding. Structure 16: 558-569.