Transgene expression in E.coli from chloroplast promoters

jon at molbiol.net jon at molbiol.net
Sun Aug 11 11:35:36 EST 2002


Bev,

Did anyone ever get a chloroplast promoter to work in E.coli?  Surely tac, lac or trp might be better...or perhaps I've missed your point.

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







http://biowww.net/mynews/tree.php?group_name=bionet.molbio.methds-reagnts&begin=0



More information about the Methods mailing list