Blunt end cloning

Cathal Garvey via methods%40net.bio.net (by cathalgarvey from gmail.com)
Thu Jul 7 06:16:35 EST 2011


I'm confused; why not try to clone the sequence into a vector *without* a
promoter first? That way you can determine whether the gene's toxicity is
all that prevents you from getting a successful transformed culture.

Alternatively, clone it with an inducible promoter. That way you can try to
establish a stable clone, and then induce expression later to test for
toxicity. It is possible that by tuning the expression with different
amounts of induction, you can find a threshold that does not kill the cells
and allows you to produce some of the gene product.

If your gene *is* toxic and you persist in trying to clone it with T7,
you'll have a hard time. Any successful transformants will be selected
against immediately, leaving only those with partial deletions or mutations
that render the gene inoperable. Try it one piece at a time and it will be
easier to isolate the problem; cloning, transformation or expression.

On 7 July 2011 00:57, mnr mnr <mnr475 from gmail.com> wrote:

> Hi Wo.
>
> It is a outer membrane protein, toxicity is probably at expression level. I
> get inserts with random deletion that I can not use for functional assays.
>
> Before I try other expression systems, I want to try other E. coli vectors
> first. Am I going in the right direction?
>
>
> On Wed, Jul 6, 2011 at 10:49 PM, WS <novalidaddress from nurfuerspam.de> wrote:
>
> > Hi Matt,
> >
> > does that mean that you don't get any clone with an insert at all or
> > "just" no clone that has a correct insert (full length, no deletions,
> > no frameshifts etc.)?
> > pUC18 is promoterless, right?
> >
> > As you suspect inherent toxicity, do you have any idea what kind of
> > toxicity it is? Will you need that toxic element or may you make it
> > "innocent" by some sort of AA substitution / motif deletion? Can you
> > possibly switch to another host like some sort of phage, YAC, (really
> > safe!!!) virus etc?
> >
> > Wo
> >
> >
> >
> >
> > On Jul 6, 7:23 pm, mnr mnr <mnr... from gmail.com> wrote:
> > > Hi all.
> > >
> > > I have tried cloning a potential toxic gene about 1.2 kbp into E.coli
> > with
> > > T7 promoter. After two months, I am still not successful. I am now
> > plannig
> > > to do cloning into pUC18 vector before subcloning into few other
> vectors
> > > with different promoters. I have limited available restriction sites so
> > that
> > > I don't have to redesign PCR primers. Is it feasible to do blunt-end
> > cloning
> > > with Phusion polymerase into non-dephosphorylated vectors? Will I only
> be
> > > getting empty vectors after transformations?
> > >
> > > Thank you.
> > >
> > > Matt
> >
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