vacuum infiltration of yeast

Preston Garrison garrisonp at THORIN.UTHSCSA.EDU
Wed Oct 26 04:37:34 EST 1994


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



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