I have used the LEU2d allele on plasmids to increase copy number and
thereby protein expression levels from LEU2d containing expression
plasmids. Plasmids that contain only the LEU2d marker are difficult to
transform and I have only been consistently successful at this when using
the spheroplast method for transformation. I believe this is because it is
necessary to introduce several copies of the plasmid into the transformed
cell in order to get sufficiently good complementation of leu2 and allow
growth on -leu plates. Possibly this problem can be reduced by allowing a
period of non-selective growth prior to plating on -leu medium. Use of a
second marker to facilitate transformation prior to imposition of the -leu
selection to drive up the copy number further works well. This way the
transformants are more stable and the leu selection can be applied only
when higher copy number is required.
Anthony Wright
Dept of Biosciences at Novum
Karolinska Institutet
Stockholm
tony.wright at cbt.ki.se
David Pearce wrote:
>>Has anybody used the Leu2-d or Ura3-d markers to get multicopy
>expression ?
>We have transformed these into strains that are very competent for
>transformation of other muticopy and CEN vectors, as well as for
>integrative transformations.
>We find that Leu2-d does not transform directly onto -leu media, even
>if left for a week as suggested in the originators paper. However, if
>another marker is present on the plasmid ( URA3 or TRP1 ), then we can
>transform onto this media and get sub-clones to grow on -leu. This
>sort of defeats the purpose that we want it for.
>The Ura3-d transforms directly onto -ura, but as the reference
>provided by ATCC does not give information on copy number, I was
>wondering if anyone had an idea, before we go ahead and check.
>>So, any previous experience with these ?
>> Dave Pearce DPEA at bphvax.biophysics.rochester.edu
