OD of saturated bacterial culture?

Duncan Clark blackhole at abuse.plus.com
Thu Sep 9 07:41:36 EST 2004


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.



More information about the Microbio mailing list