plastid promoter activity in E.coli

Johan Burger jtb at maties.sun.ac.za
Fri Aug 2 10:40:37 EST 2002


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 DH5a 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.

Does anyone have any suggestions please?

Julia Robson






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