Induction of protein expression from E. coli NEc1 - frozen cell bank to cell pellet


Abstract 

Method for inducing protein expression from NEc1, a BL21(DE3) derivative lacking slyD.  Used in the purification of 6-His tagged nanoCLAMPs. 

Instructions 

1 Start with a frozen stock of NEc1 transformed with the appropriate expression plasmid
2 Streak to single colonies on a" 2X YT Agar Plate - 2% Glucose - 100 ug/ml Carbenecillin"
3 Incubate the plate overnight at 37°C
4 Prepare an appropriate volume of starter culture medium of "2X YT - 2% Glucose - 100 ug/ml Carbenecillin" (25 ml per liter of induction culture)
5 Inoculate the starter culture with a single colony from the overnight streak plate
6 Shake the starter culture at 250 rpm for 5 hours to overnight at 37°C
7 Measure the OD600 of the starter culture
8 Prepare 1 l of induction culture medium of "2X YT - 100 ug/ml Carbenecillin" in a sterile 2.5 l Ultra Yield flask
9 Add starter culture to the induction culture medium for a final OD600 of 0.1
10 Shake the induction culture at 250 rpm until the OD600 reaches between 0.7 to 1.0 at 37°C
11 Add IPTG to 0.5 mM to induce protein expression (0.5 ml IPTG, 1 M per liter)
12 Shake the induction culture at 250 rpm for 24 hours +/- 30 min. at 30°C
13 Measure the OD600 of the induction culture
14 Weigh centrifuge bottles
15 Transfer the induction culture to pre-weighed centrifuge bottles
16 Centrifuge at 10,000 X g for 10 minutes at 4°C
17 Discard the supernatant and drain the pellet
18 Weigh centrifuge bottles and calculate the mass of the cell pellet
19 Use the cell pellet immediately or store at -70°C
The procedure typically yields 9 to 12 g cell pellet per liter

Product(s) Used
Nectagen Product Order Product
S-NEc1

Solution(s) Used
Solution Usage Rate
2X YT Agar Plates - 2% Glucose - 100 ug/ml Carbenecillin 1 plate per per strain
2X YT - 2% Glucose - 100 ug/ml Carbenecillin 25 ml per liter of induction culture
2X YT - 100 ug/ml Carbenecillin

Ingredient(s) Used
Ingredient Usage Rate
IPTG, 1 M 0.5 ml per liter of induction culture

Outside Material(s) Used
Vendor Outside Material Catalog Number Usage Rate
Fisher Scientific Ultra Yield Flask - 2.5 l 50-996-056 1 flask per liter of induction culture