Historians believe that in newspost
<c4879cbb.0409081354.4575f088 at posting.google.com> on Wed, 8 Sep 2004,
Paul Wary <paul_wary at yahoo.com> penned the following literary
masterpiece:
>I am going to express a protein in E. coli host cells (to be precise
>BL21 (DE3) cells) and the instructions say that the induction of
>protein synthesis through IPTG is to be started at an OD of 0.6, which
>is easy enough.
Standard sort of OD for shake flask IPTG inductions.
>The cells are then grown to saturation before harvesting (no OD given
>for saturation).
>Can anyone tell me what the approximate OD value of a saturated E.
>coli culture is?
It will depend upon media, type of flask (baffled or not), speed of
agitation and most importantly; how lethal or otherwise is your
over-expressed protein to the cell? If it causes problems then you may
find that your E.coli stops growing within an hour of induction. If
non-lethal you can possibly grow for 24-36 hours. The bugs will have
stopped growing but can still keep expressing. I have one that does this
and 24 hours is way better than 4 hours although the OD of the culture
is the same. Likewise I have one enzyme that is fully over-expressed
within 1 hour of induction and going beyond that does not increase the
OD or the amount of protein expressed.
I would suggest you put on a 250ml culture, take out 25ml at an OD of
0.6, add the IPTG then remove 25ml samples at 0.5hrs, 1, 2, 3, 4, 8, 16
and 24hrs.
It also wouldn't hurt to plate your innoculum (presumably an aliquot of
an overnight culture) on both antibiotic selective and non-selective
plates and cell count them. That checks that nothing nasty has happened
during overnight growth such as only 5% of the morning culture actually
contains plasmid! Usually only a problem with Ampicillin vectors and
very slight or worse lethality.
Mesaure OD at those time points, cool the cells on ice as you remove
them and when cold, centrifuge and store pellets at -7)C. Process all
pellets together and load the same equivalent sample in OD's onto an SDS
PAGE gel or assay accordingly. From that you can quickly see the best
growth/induction conditions.
Duncan
--
I love deadlines. I especially like the whooshing noise they make as
they go flying by.
Duncan Clark
GeneSys Ltd.
