Methods for transforming yeast
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 comprising the steps of:
incubating the yeast cells in a solution comprising 0.01 to 1.0 M lithium acetate (LiAc) and 1 to 100 mM dithiothreitol (DTT),
providing a suspension comprising vector DNA, insert DNA, the incubated yeast cells, 0.1 to 10 M sorbitol, and 0.1 to 10 mM CaCl 2 or MgCl 2 , and
electroporating the suspension at 5 kV/cm to 12.5 kV/cm with a capacitance of about 10 to about 50 μF;
wherein the yeast cells are transformed with a transformation efficiency greater than 1×10 8 transformed yeast cells per μg of nucleic acid vector, 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 12.5 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 the 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 comprising the steps of:
culturing 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 first solution comprising sorbitol and CaCl 2 ;
incubating the yeast cells in a second solution comprising 0.01 to 1.0 M lithium acetate (LiAc) and 1 to 100 mM dithiothreitol (DTT);
washing the yeast cells in a third solution comprising sorbitol and CaCl 2 ;
resuspending the yeast cells in a fourth solution comprising 0.1 to 10 M sorbitol and 0.1 to 10 mM CaCl 2 or MgCl 2 to form a yeast cell electroporation suspension;
adding a volume of the yeast cell electroporation 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 5 kV/cm and about 12.5 kV/cm in a 0.2 cm gap cuvette,
wherein the yeast cells are transformed with a transformation efficiency greater than 1×10 8 transformed yeast cells per μg of nucleic acid vector, thereby to obtain a library size of at least 1×10 9 .
14. The method of claim 1 , wherein the yeast belongs to species Saccharomyces cerevisiae.
15. The method of claim 13 , wherein the yeast belongs to species Saccharomyces cerevisiae.