The EasySelect Pichia expression manual by Invitrogen says zeocin is not needed after clone selection, but when we express with zeocin we get lots of protein production and when we express without zeocin there is no protein production. Has anyone else had this happen or know why this is happening?
Melissa,
Is the protein toxic to the cells or slowing down growth? So without antibiotic pressure, non expressing mutants might overgrow the culture.
Have you tried to rescue the cDNA from isolated colonies and sequence it, also its promoter regions?
I assume that it can only happen if the vector is not integrated with the Pichia genome or integrated and then removed. Therefore the ZeocinR-vector-insert is loosely floating in the cell and if you did not give the antibiotic pressure it loses the copy number (!?).
see if the gene is integrated in the genome using PCR may be (consult the manual for integration portion of the vector).
I select with zeo, then express without Zeo. However, I have screened clones (2ml), with zeo and methanol at the same time. Unfortunatly, i have never done a side by side comparison
The vector we used is a suicide vector so it cannot be replicating in the cell. We checked that the gene was integrated with the suggested primers from the manual and it was positive. We attempted expressing with lower zeocin concentrations (25 µg/mL instead of 100 µg/mL) and expression was good. But when zeocin is omitted completely we get no expression.
Hi, I wonder if the buffered minimal or complex media is compatible with zeocin. My concern is that phosphate can tightly bind to the copper of the zeocin thus inactivating it. In addition, yeast nitrogen base (YNB) is mostly ammonium sulfate, and it is there at 1.34%, which gives comparable ionic strength to the standard LB with 1% NaCl, and it is said that only low salt LB can be used with zeocin with E.coli. Maybe the ionic strength is not applicable to pichia
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