Design of expression plasmid

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


In article <920721161723.21400d77 at ntet.nctr.fda.gov>, 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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