On Jan 6, 2:28 pm, "Boettcher, Tara" <dac... from ll.mit.edu> wrote:
> I am using CC-125 and CC-124 and trying to transform them with pSP124S with Bleo resistance marker, using both Karen Kindle method with glass beads and the Purton variation of the Karen Kindle method, and not having any luck.
> I've done an agar kill curve with zeocin, I have linearized the plasmid, and have tried autolysin treatments.
>> Does anybody have insight on 'crucial steps', a new method to try, or a new plasmid with a different resistance marker I could try?
> Any help is greatly appreciated!
I have been transforming Chlamydomonas D66 with the sSP124S plasmid
because my background mutant already has the Streptomyces rimosus
aphVII (pSI103 plasmid with a aminoglycoside phosphotransferase
conferring paromomycin resistance) marker (Sizova et al. 2001). You
can readily get this plasmid from the Chlamy Center (http://
www.chlamy.org/plasmids.html) and it seems to be a marker that is much
easier to use.
As I understand, the BleR marker has fallen out of favor because of
some of the difficulties that can occur. For one, there seems to be
different strengths of Bleocin/Zeocin from batch to batch and each
order should be titrated separately. Further, the Sizova paper
mentions that the BLE protein dimer can only inactivate two antibiotic
molecules through drug sequestration. So it is as if the BLE
resistance is quenching the drug. Thus, variable expression levels in
your mutants should confer a pool of variable resistance in the
population of mutants you plate. If your concentration of Ble is too
high, you will select for mutants with mulitiple resistance gene
insertions. I have also noticed variable susceptibility to the zeocin
antibiotic in the wild-type cells based on factors like culture growth
stage and, of course, the density of cells plated. When working with
Ble-resistance it is important to have a good negative control that
goes through the entire transformation process, but without the
introduction of the plasmid. This is important, because the current
electroporation method I am employing makes even the wild-type cells
more susceptible to bleocin. I suspect, but have not tested, that
this is due to a combination of diluted cell number during transfers
and cell mortality during electroporation. Zeocin is also a mutagen,
which can result in point mutations unrelated to your site of
insertion, making tracking down the cause of a mutant phenotype nearly
Microscopy confirms that the bead transformation methods lyses many
cells. As with your experience, I have had poor transformation
efficiency with the bead method, but it does work. It is important to
try lots of variations and keep records of everything (including cell
density at start of experiment ect.). I have also had good luck with
increasing the the plasmid concentration to make the bead beating
method work, but this has been reported to also increase the number of
transformants with multiple insertions. You can also try plating the
cells with starch.
Overall, you will likely have the best luck with the pSI103 plasmid
and electroporation. The APH protein inactivates paromomycin by
transferring a phosphate from ATP to the paromomycin antibiotic, and
can keep catalyzing this reaction. Thus, the effective range of
paromomycin, paromomycin:cell ratio, and variability due to
variability in gene expression/gene copy number reasoned to be much
better. In my hands, this seems to be the case.
Hope this helps!