Design of expression plasmid

Michael Benedik bchs1b at Elroy.UH.EDU
Wed Jul 22 21:59:51 EST 1992

In article <920721161723.21400d77 at>, WMELCHIOR at NTET.NCTR.FDA.GOV (Bill Melchior, NCTR/FDA) writes:
>I am considering putting a gene under lac control in pBR322, using the lac
>promoter from pOP95-5 (F. Fuller, Gene 19, 43-54 (82)).  I have a question
>(actually several, but I won't bother you with the others):  Fuller says that
>the gene should be placed "near" the 3' end of the promoter, but he doesn't
>say what "near" means, and I haven't been able to find out from any of the 
>other sources I've consulted.  Does anyone know what the acceptable range
>is for distances between, say, the "-10" region of the promoter and the
>Shine-Dalgarno sequence or the initiation codon?  (My gene has a native S-D 
>sequence and an initiation codon.  I'm not making a fusion protein.)  I 
>don't need super efficient expression, but do need some.
>Thanks, Bill
>The opinions stated are mine, not those of NCTR or its sponsoring organizations.
>Bill Melchior                                ||   "You have lawyers the way
>National Center for Toxicological Research   ||    other people have mice."
>Jefferson, AR  72079                         ||
>(501) 543-7206                               ||   -Kenneth Duncan, English
>                                             ||    Health & Safety Executive,
>WMELCHIOR at NTDOC.NCTR.FDA.GOV                 ||    to US regulators

It really depends upon what the sequence is in the spacer region. But you
do have the phenomenon of polarity in E.coli caused by transcribing regions
which are untranslated. A reasonable guess would be:
500 bp will probably be ok, but not always
100 bp should almost always be fine for reasonale expression
less than 50 probably ideal

	 Michael Benedik
	 Department of Biochemical and Biophysical Sciences
	 University of Houston
	 INTERNET: Benedik at UH.EDU	BITNET: Benedik at UHOU

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