newera at plaza.snu.ac.kr () wrote:
>Why is the temperature control for growing E. coli under the T7 RNA polymerase/promoter system required?
>>Page 16-6 in "Short Protocols In Molecular Biology 3rd ed. edited by Fred Ausubel et al." explains the procedures for growing E. coli under the T7 RNA polymerase/promoter system :
>>"...
>5) Pick a single E. coli... Inoculate it into 5ml LB/ampcillin/kanamycin medium and grow overnight at *30 degrees C.*.
>6) Dilute 1ml 1:40 into LB... and grow several hours at *30 degrees C.* to an OD = 0.4.
>7) Induce gene by quikly raising temperature to *42 degrees C.* for *30 min*.
>8) Reduce temperature to *37 degrees* C. and grow cells an additional *90 min*... Harvest.
>..."
>>I would like to know why the temperature is controlled like the above.
>>Is it a must to grow E. coli efficiently under the T7 RNA polymerase/promoter system and can it be also applied to M9 medium?
>Does it prevent inclusion body or insoluble protein aggregate formation?
>>Any comments will be appreciated.
>>Thanks,
>>Lee, Ji Hyun
>>--
>email : newera at plaza.snu.ac.kr>address :
> Lee, Ji Hyun
> Laboratory of Physical Pharmacy(Prof. Lee, Bong Jin)
>> Seoul National University
> College of Pharmacy
> Shinlim-Dong, Kwanak-Gu
> Seoul 151-742, Korea.
>
The short anwser to your question is that the expression of T7 RNA Pol
on the plasmid you are using is controlled (probably) by the
temperature sensitive cI857 promoter. This is a promoter which at
temperatures below 35C should not allow expression of the gene it is
controlling due to the binding of a temperature sensitive protein.
Above 40C the temperature sensitive protein which prevents expression
denatures and transcription is possible. E. coli does not survive
well at 42C, hence the 42C is only long enough to enable production
of sufficient transcript to produce T7 RNA Pol and then the
temperature is reduced to 37C so that E.coli can get on with its life!
There are lots of caveats to the above, such as the fact that there
is some expression from this type of system even at 35C.
Hope that this helps
gordon
*******************************************************
Gordon Munro email: g_munro at globalnet.co.uk
Cytocell Ltd
Banbury
UK
visit our website: http://www.cytocell.co.uk
Opinions expressed are my own and not necessarily those of Cytocell Ltd.
************************************************************************
