plates before stress to serve as reference 100 survival in total 8 cycles of freezing in liquid nitrogen for 30 seconds and thawing for 3 minutes in water bath at 30 u00b0 c followed by plating of aliquots on ypd plates were carried out survival was scored by counting colony forming units cfus it has previously been shown that wildtype strains can withstand the stress of freezing and thawing better than aqua porin deletion strains tanghe et al 2002 the reduced number of cfus in the aqy1 deletion strain proves that aqy1 was successfully disrupted spheroplast assay to confirm reduction of water transport in aqy1 deletion strain pichia pastoris cells both wildtype x33 cells and the aqy1 deletion strain were grown for 24 h in 10 ml bmgy in a rotary shaker at 220 rpm and 30 u00b0 c subsequently the protein production was induced by growing the cells in 10 ml bmmy for 24 h as above starting at od 600 1 by spinning down the respective amount at 3000 xg 10 min 20 u00b0 c the cells were sedimented at 3000 xg for five minutes at 20 u00b0 c and resuspended in 1 4 ml g wet cell weight of te buffer 100 mm tris ph 8 0 100 mm edta and diluted to a final volume of 3 5 ml g wet cell weight with dh 2 o 2 mercapto ethanol 1 200th of volume wet cell weight was.