Transgene expression in E.coli from chloroplast promoters

Michael Witty mw132 at mole.bio.cam.ac.uk
Wed Jul 24 07:14:22 EST 2002


On Wed, 24 Jul 2002, Bev wrote:

> I have a construct that includes a promoter for the ribsomal RNA operon, of
> grape chloroplast origin, followed by a short MCS, the aadA gene conferring
> spectinomycin resistance (or BADH), a ribosome binding site GGAGG, 9
> nucleotides upstream of the initiation codon, the GFP gene, and the psbA
> terminator, also of grape chloroplast origin.  I would like to test this
> system in E.coli, using the grape chloroplast promoter (16Srrn) to drive the
> expression of the GFP gene.  The region from promoter to terminator was
> cloned into a modified plasmid, where the T3 and T7 promoters had been
> eliminated to enable an unambiguous test for activity of the chloroplast
> promoter.  (A negative control was also constructed, excluding the
> promoter).  I cloned the constructs into DH5? ultra competent cells and grew
> them on both Luria and Terrific Broths.  We have not been able to visualize
> any GFP expression in E. coli.
>
> Any suggestions please?
> Thanks

Dear Bev,
        have I got this right: the rrn promoter drives the aadA gene as
well as the GFP gene?  Are your E. coli streptomycin resistant?  If so,
then at least your grape chloroplast promoter is OK.  Mike.




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