Superbroth for EVERYONE! (was NOT, but now IS)

Ted M. tedm at darkwing.uoregon.edu
Wed Feb 28 01:12:40 EST 1996


In article <horowicz-2702961947360001 at mac056027.lif.icnet.uk>,
horowicz at icrf.icnet.uk (David Ish-Horowicz) wrote:

> I already got four requests to post the text version in 2 hours, so here
> it is. Acknowledgements to David Micklem and Gustilo for forwarding it to
> me, and to Dave Smith for posting it (or trying to) in the first place. No
> figures.
> 
> David Ish-Horowicz
> 
> 
> 
> SUPER BROTH APPARATUS AND PROTOCOL

>            Super Broth Media Recipe:
>      
>      32 g Tryptone
>      20 g Yeast Extract
>      5 g  NaCl
>      5 mL 1.0 M NaOH
>      
>      Bring to 1 liter with house distilled water.
> 
> Typical Results
> 
> Under the conditions described the host E. coli BL21 (DE3)
> harboring the plasmid pET11aS105 inoculated at an initial OD550 of
> 0.025 requires 13 hours to reach an OD of 100 (see Fig. 2).
> 
> David Ish-Horowicz
> Imperial Cancer Research Fund, London, England
> horowicz at icrf.icnet.uk


Much edited...
   Congrats David you've built a fermentor, kind of... You may know that a
similar idea has been floated many times as a method to produce plasmids
etc with a minimal space in the lab, there are even some older flasks with
sparge stones sitting forgotten down the hall from me from yeast work in
the 70's. I seem to remember a company selling little units with fish
pumps which they promoted as the most space efficient way to produce
plasmids, vs shake flasks & shakers etc. 
   I do find it hard to belive that you can get an OD anywhere near 100
even with 13h of growth. The real hurdle is the media, it doesn't have
enough stuff. Without a readily available carbon source such as bolus
glycerol or fed glucose, you can't get cell yields like that! An OD550 of
100 corresponds to roughly 50grams/L of E. coli, albeit wet weight, but I
don't think the mass balance can be that efficient, given there is only
50grams of anything to begin with. Maybe all the years I spent running
fermentors with fancy glucose/salts/nitrogen source feeds where really
more misspent than I already suspect, but I hope not! Aside from my
disbelief, I think can offer some insights into whats going on. 
     You are right to attempt to improve std molecular biological methods
of growing bugs; they are generally very primative. Many labs don't even
use baffled flasks or rich media, much less fermentors, in protein
inductions. Perhaps it is because of the added bother, or because going
through 12 doublings (as in your case) will bring to light any plasmid
instability your construct may have. Keep in mind that about as soon as
the media is turbid the antibiotic is GONE; no selection. Further, without
good buffering (or pH control) any glucose present will likely be shunted
through the bacterial Crabtree pathway and make lots of organic acids,
because of incomplete aeration. The pH can dump and kill the cells etc.
Some E. coli make a lot of acid (MC1061) while others make very little
(JM105). Usually hyperoxygenation is not the complete answer (put away the
O2 tanks) what works best is to feed the sugar gradually so as to maintain
fast growth and not tox out the cells. If the cells run out of readily
available carbon source (or never had any as in the SuperBroth) they will
activate carbon salvage from aminoacids and the pH actually goes UP,
ostensibly because of stripped amines basifying the media, also not good. 
   To your defense, David, you got two things right on! Aerate the bugs
and feed them nitrogen source. I'd add two more things: give them carbon
source and buffer them. Some useful additions to the Superbroth would be
buffering salts; minimally 1X M9 salts, or a nice phosphate buffer around
pH 7. Secondly give them a good carbon source which isn't as tricky as
glucose: bolus glycerol. Adding 5-10 grams per liter initially would give
higher cell yields. If after the longer growth in you bubbler the pH isn't
bad, the cells are viable for plating, some plasmid remains and your
protein looks good, by all means enjoy the increased cell yield and spend
the saved time doing more compelling science.

regards,
Ted Michelini
Institute of Molecular Biology
University of Oregon
tedm at darkwing.uorgon.edu



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