vacuum infiltration of yeast

[Stan Du]

> Kevin Piers wrote:
> >Hello oh fellow yeasters,
> >
> >I have a question that may initially sound bizarre, but I am relatively 
> >new in this field.  I am trying to establish a system by which I can 
> >routinely and consistently transform yeast using agrobacterium as a donor 
> >organism.  I have all the appropriate vectors set up and have been able 
> >to obtain transformants in this way.  Due to the fact that S. cerevisiae 
> >is relatively "non-sticky" and that cell to cell contact is necessary for 
> >DNA transfer in this system, I have promoted such interactions by vaccuum 
> >infiltrating these organisms together.  That is, once they are mixed and 
> >place on a filter, I subject them to a time in a vacuum. This treatment 
> >results in a higher transformation frequency.  Now, FINALLY, the 
> >question:  Has anyone ever subjected yeast to vacuum infiltration and if 
> >so, under what circumstances and what effects did it have on the cell.  
> >Although I am running the proper controls, I am slightly concerned about 
> >the effects of such a treatment on the yeast cells.  Any information 
> >would be greatly appreciated and can be sent directly to me.  Thanks in 
> >advance.
> >
> >Kevin Piers
> >Dept. Microbiology
> >University of Washington
> >Seattle WA 98195
> 
> What sort of effects are you worried about? Collecting yeast on a filter 
> by vacuum is a fairly standard procedure for getting them out of 
> liquid culture quickly, as for nucleotide measurements. Normally one doesn't 
> keep them on the filter for a long time, although Susan Moore used to do
> experiments where she put a monolayer of cells in agar on a filter and 
> perfused it with medium by pulling a vacuum on it (Exp Cell Res 171: 411 
> 1987 and refs therin). Yeast are tough little critters - a little bit of 
> pressure isn't going to hurt them. If you put them in a thick layer the ones
> underneath may get O2 starved, but only if they are in respiratory mode. If
> they have been on glucose they won't be using much O2 anyway.
> 
> Hope this helps,
> 
> 
>  
> 
> Preston Garrison                 garrisonp at uthscsa.edu
> Biochem. Dept.                   voice: 210-567-3702
> Univ Texas Health Sci Ctr        fax:   210-567-6595
> San Antonio, Tx 78284-7760
> USA