sd_labber

Hi all,

I've been working with the GS115 strain of P. pastoris for the past month.  I'm using Invitrogen's EasySelect kit, and I did not have any issues with the cloning or selection (all clones are zeocin-resistant and show desired inserts).  In the beginning I was working with the X33 strain and had no issues with growth or expression, then my cells suddenly wouldn't grow well in MGY media, only YPD.  Even when I inoculated MGY with a strong YPD culture of the yeast the growth was weak.  I switched strains to GS115, proceeding from scratch, and I see the same issue.  It's been 4 days since I picked colonies from zeocin plates into MGYH media, and the cultures are very weak and smell sour.  I've tried varying glycerol, YNB, and biotin sources to make sure I wasn't using a bad reagent, and I keep the shaker set at 220rpm, 28 degrees.  Is it an issue with pH or some other factor?

Any advice?  I need strong MGY/MGYH cultures to get our fermenter going.  Thanks!