Difference: ExpressMediaM9 (27 vs. 28)

MATERIALS

REAGENTS

• Ampicillin or carbenicillin
• Plasmid pRSF.1b (Novagen), containing the cDNA for the protein
• M9 medium (see REAGENT SETUP)
• M9 salts: M9 medium without the addition of NH₄Cl,MgCl₂, thiamine, glucose or glycerol etc
  
• Escherichia coli BL21(DE3) CP RIL (Stratagene, Cat No. 230245) BL21(DE3) plys (Novagen, Cat No. 70236-4) BL21(DE3) (Novagen, Cat No. 70235-4)
• 0.5 M IPTG
• LB medium and plates (see REAGENT SETUP)
• 10 mM potassium phosphate buffer (pH 7.0)
• Vitamin Solution: KAO AND MICHAYLUK VITAMIN SOLUTION (100X) Sigma (Cat No. K3129)

REAGENT Setup

LB medium and plates For 1 l, combine the following:

• 10 g peptone from casein
• 5 g yeast extract
• 5 gNaCl

M9 medium For 1 l, combine the following in double distilled or 16 Mohm water :

• 6.2 g Na₂HPO₄
• 3 g KH₂PO₄
• 0.5 gNaCl

• 1 g NH₄Cl or [U-¹⁵N]NH₄Cl
• 2 ml 1 MMgSO₄.7H₂O
• 1 ml 1 M CaCl₂.5H₂O
• 10 ml Vitamin Sol
• 1ml 100 mg/ml ampicillin or similar
  
• 10 ml 20% (wt/vol) D-glucose or 2-3 g [U-¹³C]glucose
• 1 ml Metal Sol-I (see end)
  
• 1 ml Metal Sol-II
  

PROCEDURE :

1. Transform E. coli strain BL21 (DE3), or its derivatives, with pRSF.1b expression plasmid(s)
2. Grow the transformed cells on an LB plate containing 100 mg/ l ampicillin (LB-Ap). ▲ CRITICAL STEP Glucose (0.2%) may be added to the LB plate to suppress any leakiness in the araBAD promoter if used (PBAD). Glucose reduces the concentration of cAMP in these cells, preventing cAMP-binding protein (CAP) from activating transcription.
3. Inoculate 500 ml LB-with antibiotic(s) with a single transformed colony and grow the cells overnight at 37 °C with shaking to a maximum OD 600 of ~1.6. ▲ CRITICAL STEP Growing the culture to a higher OD results in the loss of the ampicillin resistance plasmid by the bacteria (because ampicillin is secreted into the growth medium) and the subsequent inability to express the protein of interest. This may be overcome by using carbenicillin (150 mg/ 1) instead of ampicillin. For very high level of incorporation of isotopes, consider the initial growth in M9 (will be slower by 2x or more).
   
4. Centrifuge (~5000 g, 5 min, 4 °C) enough cells to yield ~0.5 OD600 in 1 l.
5. Wash the cells once with 500 ml M9 salts and resuspend in 1 l M9 medium containing nitrogen and carbon sources, and 100 mg/l ampicillin
6. Remove 1 ml of uninduced cells for analysis by SDSPAGE. Pellet the cells for 1 min at maximum speed at room temperature using a benchtop centrifuge, withdraw the supernatant and freeze the cells at –20 °C.
7. Incubate the culture at 37 °C for 10 min and induce protein overexpression by adding 0.1% culture volume of 0.5 M IPTG.
8. Allow the production of labeled target protein to proceed for 4 h. Remove 1 ml of induced cells for analysis by SDSPAGE; process the cells as described in Step 6.
9. Measure the OD600 of the culture and centrifuge enough cells to yield an optical density of ~0.5 OD600 in 300 ml.
10. For high levels of expression , the 4 h sample can be harvested. In case of low levels of expression assayed by SDS Page, it may be necessary to consider,
    1. lowering temperature after induction and expressing for a longer period
       
    2. inducing in several stages, seperated by 2-3 h.
11. Harvest cells by centrifugation , and break using preferred method (French Press, or sonication etc).
    

Metal Solutions

-- DavidCowburn - 14 Apr 2008