Temperature control in the T7 RNA polymerase/promoter system. (For protein expression)?

newera at plaza.snu.ac.kr newera at plaza.snu.ac.kr
Wed Feb 26 01:00:44 EST 1997


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.




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