Methods for transforming yeast

Abstract

This invention is directed to the transformation of yeast, and mutants thereof, by electroporation, which result in stably transformed yeast host cells that express recombinant products. This invention also is directed to transformed yeast cells and libraries.

1 . A method of preparing a yeast library by electroporation of yeast cells, the method comprising the steps of:
 providing a suspension comprising nucleic acid vectors, yeast cells, sorbitol, and CaCl 2  or MgCl 2  and   electroporating the suspension at 0.5 kV/cm to greater than 12.5 kV/cm with a capacitance of about 10 to about 50 μF; thereby to obtain a library size of at least 1×10 9 .   
     
     
         2 . The method of  claim 1 , wherein the suspension comprises 1M sorbitol and 1 mM CaCl 2 . 
     
     
         3 . The method of  claim 1 , wherein the suspension comprises 1M sorbitol and 1 mM MgCl 2 . 
     
     
         4 . The method of  claim 1 , wherein the yeast cells are incubated in a volume comprising cold 0.1M LiAc and 10 mM DTT. 
     
     
         5 . The method of  claim 1 , wherein the suspension is electroporated at 2.5 kV/cm with a capacitance of about 25 μF. 
     
     
         6 . The method of  claim 1 , wherein the electroporating step is performed in a 0.2 cm gap cuvette. 
     
     
         7 . The method of  claim 1 , wherein 1 μg to 8 μg of vector DNA is used. 
     
     
         8 . The method of  claim 1 , wherein 4 μg of vector DNA is used. 
     
     
         9 . The method of  claim 1 , wherein vector is linear. 
     
     
         10 . The method of  claim 1 , wherein the suspension comprises 400 μl of 1.6×10 9  yeast cells/mL. 
     
     
         11 . The method of  claim 1 , wherein the ratio of vector DNA to insert DNA is in the range of 1:0.5 to 1:10. 
     
     
         12 . The method of  claim 1 , wherein the ratio of vector DNA to insert DNA is 1:3. 
     
     
         13 . A method for transforming yeast with DNA to prepare a library, the method comprising the steps of:
 Culturing an over night yeast cells to an OD 600  of about 1.0 to about 2;   Washing the yeast cells in water;   Washing the yeast cells in a solution comprising sorbitol and CaCl 2 ;   Incubating the yeast cells in a solution comprising LiAc and DTT;   Washing the yeast cells in a solution comprising sorbitol and CaCl 2 ;   Resuspending the yeast cells in a solution comprising sorbitol and CaCl 2  to form a yeast cell electroporation suspension;   Adding a volume of the electroporation cell suspension to vector DNA and insert DNA to form a yeast cell—DNA electroporation suspension; and   Electroporating the yeast cell—DNA electroporation suspension at a voltage of between about 2.5 kV/cm and about 12.5 kV/cm in 0.2 cm gap cuvette.