Materials
Expression plasmid (e.g.,pET/Ncd in BL21(DE3)pLysS host cells)
ZB = 10 g NZ-Amine A
5 g NaCl
860 ml final volume
10X M9 Salts = 1 g NH4Cl
3 g KH2PO4
6 g Na2HPO4
100 ml final volume
M9ZB = 860 ml ZB
100 ml 10X M9 Salts
40 ml 10% glucose
1 ml 1 M Magnesium Sulfate
1 ml 50 mg/ml Ampicillin in DDW
0.1 ml 50 mg/ml Chloramphenicol in EtOH
Sterile glycerol (0.5 ml in sterile tube)
1 M IPTG,dioxane-free in DDW
200 mM PMSF in EtOH
Special Equipment
Refrigerated Incubator Shaker (e.g.,New Brunswick Innova 4330)
Procedure
1.Inoculate 1 ml of M9ZB with single colony from newly transformed cells or freshly streaked plate from glycerol culture of newly transformed cells.
2.Grow cells for 1.5 hr at 37℃.
3.Inoculate 20 ml M9ZB with the 1 ml culture and grow 1.5 hr at 37℃.
4.Inoculate 4 x 500 ml M9ZB in 2 L culture flasks with 5 ml of the culture and grow overnight (12-14 hr)at 22℃ to OD550 = 0.9 to 1.0.
5.Sterilely withdraw 0.5 ml cells,mix with 0.5 ml sterile glycerol in a sterile tube,freeze at -80℃ for future use as stock.
6.Add IPTG to 0.4 mM to induce protein expression and shake at 22℃ for 3.5-6 hr.
7.Harvest cells by swirling in ice water bath for 5 min,transfering to centrifuge bottles on ice and centrifuging at 6,500 rpm (7,100 x g)in a Sorvall GS-3 rotor.
8.Resuspend cells in column buffer containing 0.5-1 mM PMSF and centrifuge in 30 ml tared Oak Ridge tubes.
9.Weigh tubes,should contain 2-5 g cells/tube.
10.Flash freeze in liquid N2 and store at -80℃ until use.
Notes
1.The use of newly transformed cells or a freshly streaked plate from a glycerol culture of newly transformed cells improves protein expression.
2.To optimize induction time,grow a 20 ml culture to OD550 = 0.9 to 1.0,withdraw 0.5 ml for 0 timepoint,then induce by adding IPTG to 0.4 mM and take 1 hr,2 hr,4 hr,6 hr and overnight timepoints.Centrifuge samples after collection for 1-2 min in a microfuge to collect cells and resuspend pellet in 30 microliters 1X SDS loading dye solution.Analyze by SDS-PAGE (5-10 microliters/lane)to determine the optimal time of induction.
3.To monitor induction of large cultures,withdraw 0.5 ml of cells from each flask just prior to induction and just prior to harvest.Centrifuge samples for 1-2 min in a microfuge and resuspend pellet in 30 microliters 1X SDS loading dye solution.Analyze by SDS-PAGE to determine if the cells are induced.
4.For biotinylated motors,grow cells in M9ZB media containing 1 µM biotin and add biotin to 2 µM at the time of induction.
5.Plasmids may be lost from cells during growth or induction.It is therefore necessary to test each batch of induced cells for protein expression.
