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 <[email protected]> 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 <[email protected]> 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 <[email protected]> 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 > > > > _______________________________________________ > > Methods mailing list > > [email protected] > > http://www.bio.net/biomail/listinfo/methods > > > _______________________________________________ > Methods mailing list > [email protected] > http://www.bio.net/biomail/listinfo/methods > -- letters.cunningprojects.com twitter.com/onetruecathal http://www.indiebiotech.com _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
