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coli cells
IMPORTANT:
For the best control for your induction: transform the vector only (without your gene)
into the bacteria, and take one sample of each induction condition for comparison of
induction on coomassie.
1. Transform the plasmid DNA into the appropriate bacterial strain For example:
for pET vectors:(BL21(DE3)pLysS or BL21(DE3)
You can use this starter for additional induction experiments for about 10 days.
You may want to keep a glycerol stock that might be good for a few months from this growth.
However, in most clones, you can lose the expression after a few weeks/months, and you will
have to start from a fresh de-novo transformation of DNA into the expressing bacterial strain.
3.Add 1.2 ml starter into 120ml 2xYT medium containing the antibiotics and grow at 37oC
till OD600=0.6
4.Remove 2 x 1.5 ml of bacterial cells into sterile tube –on ice (un-induced
sample), spin down aspirate sup and freeze pellets in –20C
7.Add IPTG to the 37oc flasks to final concentration of 0.1 0.4 and 0.8mM. (See
notes below for Glucose suppression)
8.Let the 23 oc flasks shake for 15 minutes. Then add IPTG(0.1 0.4 and 0.8mM)
to these flaskstoo.
9. Grow on shakers at 225rpm and take 2-3 x 1.5 ml samples from each
pellets at -20ºC
For further analysis of your samples, lysis is required. This protocol includes sonication.
Alternative procedures are
B-per detergent (for initial screening of several samples)
This screen may show that most of your protein is found in the pellet, whereas following lysis by
sonication your protein may be found in the sup.)
French-press
(The bacterial concentration proportional volumes of protein samples are indicated by the OD
measurements of the bacterial suspension at each time point before the cenrifugation at step 9 in
the first protocol).
When proteins are sensitive to heat shock, try: De Marco A, Vigh L, Diamant S,
Goloubinoff P. Native folding of aggregation-prone recombinant proteins in Escherichia coli by
osmolytes, plasmid- or benzyl alcohol-overexpressed molecular chaperones. Cell Stress
Chaperones. 2005, 10(4):329-39
2.Add 0.1% glycerol and 0.1mM Potassium Glutamate directly into the
medium (We use: SIGMA G-1501 L-Glutamic acid monopotasium salt)
3.Heat shock for 20-30min at 42oC
4.Transfer to original temperature (23oC or 37oC) and let cells grow to OD 0.7-
0.8 .
6.Follow same induction procedure as before (pick samples after 2h/4h/on etc.)
Notes:
If suppression of the expressed protein is not complete, and the proteinis toxic to the cells or
forms inclusion bodies, one can grow the DE3 based bacterial strains in the presence of 1%
glucose to suppress basal expression. In this case, another calibration of IPTG induction is
required, with higher amounts of IPTG (0.5mM, 1mM and 2mM), since the effective
concentration of the IPTG is now lower.