Fresh transformation of BL21; how?

Troels Wind wind at biobase.dk
Thu Dec 14 11:25:16 EST 2000


Hi there,

Like others in this newsgroup I've observed clone-to-clone variations for
BL21(DE3)pLysS transformants with pET derivatives, but what I see is not
different levels of expression but different levels of growth rate. In
short, after transformation and plating, I inoculate a few overnight
cultures at 30C from individual colonies. The next day, I dilute these 1:50,
follow their growth at 37C and observe that some cultures grow "fast", i.e.
reaches OD ~0.8 in <3 h while others need >6 h for this. From the fast
cultures I can purify >5 mg of soluble recombinant protein, so it dosnt seem
to be toxic. The slow cultures also produce my protein in good amounts upon
induction.

What I will try now is to avoid bringing the cultures to stationary phase to
see if I can get reproducible growthcurves, but I would really appreciate it
if someone would share their experience with this. Do you skip the plating
of your transformation and inoculate in liquid culture straight away? Or do
you inoculate colonies and then wait for them to reach OD 0.8 (or whatever
OD you induce at)? In either case, whats the conditions in terms of broth (I
use 2xTY), temperature, and whats the time-scale?

Thanks in advance,
Troels


Troels Wind, Ph.D.
Laboratory of Cellular Protein Science
Department of Molecular and Structural Biology
University of Aarhus
Gustav Wieds Vej 10C
8000 Aarhus C
Denmark

Phone: (+45) 8942 5079
Fax: (+45) 8612 3178
Mobile: (+45) 2856 9767

"Only two things are infinite, the universe and
human stupidity, and I'm not sure about the former."
                                                   -Albert Einstein


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