Overexpression

Enrique Jose Labadan Frio - SCBC - 3421101 g3421101 at MUCC.MAHIDOL.AC.TH
Tue Feb 22 04:30:09 EST 1994


> A special problem seems to be that expression of our gene is lethal to 
> the E. coli host so we need a system based on a promotor under strong 
> control.  
	
	You can try the T7 RNA polymerase/promoter system by S.Tabor (1990)
in Current Protocols in Molecular Biology (pp 16.2.1 - 16.2.11).

	The system consists of a T7 RNA polymerase (highly processive and
does not require special transcription factors since it is viral) under
the control of the lac promoter. The polymerase is produced under heat
induction (42 oC) by the inactivation of a heat-sensitive lambda repressor
(mutant) bound to the polymerase's lac promoter.

	The system has another plasmid for cloning in your fragment (3
types actually, with different multiple cloning site configurations,
including one with a strong ribosome binding site for expression), under
the control of the T7 promoter. The cloning plasmid, when co-transformed
with the RNA pol plasmid, gives a good expression pair: the T7 polymerase
transcribes only from its promoter (thus, the gene you've cloned) and goes
on very rapidly, producing much mRNA transcript. 

	To control the expression, one just dips the whole flask into a 
cooler water bath to bring the temperature down to 30 oC, where the 
mutant repressor shuts the RNA pol promoter.

	Good luck.. let us all know of your progress!

Querix Frio
Graduate student

E-mail: g3421101 at mucc.mahidol.ac.th	






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