Method for lyophilizing competent cells

Abstract

This invention relates to a method for producing cells which are competent for transformation and which may be stably stored for extended periods of time at various temperatures. The method involves growing cells in a growth conducive medium, rendering said cells competent, and lyophilizing said competent cells. The invention further relates to competent cells produced by such a method, to methods of transforming said cells with a DNA molecule, and to a method of producing a desired protein or polypeptide from said transformed cells.

1 . A method for producing cells which are competent for transformation, said method comprising
 (a) growing said cells in a growth conducive medium;   (b) rendering said cells competent; and   (c) lyophilizing said competent cells.   
   
   
       2 . The method of  claim 1 , wherein said cells are grown at about 10° C. to about 42° C. 
   
   
       3 . The method of  claim 1 , wherein said cells are grown at about 12° C. to 37° C. 
   
   
       4 . The method of  claim 1 , wherein said cells are grown at about 15° C. to about 32° C. 
   
   
       5 . The method of  claim 1 , wherein said cells are grown at about 20° C. to about 25° C. 
   
   
       6 . The method of  claim 1 , wherein said cells are grown at about 23° C. 
   
   
       7 . The method of  claim 1 , wherein said cells are gram negative prokaryotic cells. 
   
   
       8 . The method of  claim 7 , wherein said cells are selected from the group consisting of  Escherichia, Klebsiella, Salmonella , and  Pseudomonas.    
   
   
       9 . The method of  claim 8 , wherein said cells are  Escherichia  cells. 
   
   
       10 . The method of  claim 9 , wherein said cells are  E. coli  cells. 
   
   
       11 . The method of  claim 10 , wherein said  E. coli  cells are selected from the group consisting of DH5, DH5α, DH10, DH10B, HB101, RR1, JV30, DH11S, DM1, DH10B/p3, DH5αMCR, DH5α5′IQ, DH5α5′, SCSI, Stab2, DH12S, DH5α-E, DH10BAC, XL1-Blue MRF, XL2-Blue MRF, XL1-Blue MR, SURE Strain, SURE 2 Strain, XL1-Blue, XL2-Blue, AG1, JM101, JM109, JM110/SCS110, NM522, TOPP Strains, ABLE Strains, XL1-Red, BL21 Strains, TK B1 Strain, and derivatives thereof. 
   
   
       12 . The method of  claim 1  wherein said lyophilizing step comprises
 (a) freezing the cells; and   (b) subjecting said frozen cells to a vacuum under conditions sufficient to substantially dry said cells.   
   
   
       13 . The method of  claim 1 , wherein said step of rendering said cells competent comprises suspending the cells in a competence buffer. 
   
   
       14 . The method of  claim 13 , wherein said competence buffer is CCMB80 buffer. 
   
   
       15 . The method of  claim 1 , wherein said method further comprises mixing said cells with a cryoprotectant prior to the lyophilizing step. 
   
   
       16 . The method of  claim 15 , wherein said cryoprotectant is a carbohydrate or derivative thereof. 
   
   
       17 . The method of  claim 16 , wherein said carbohydrate is selected from the group consisting of trehalose, sucrose, sorbitol, glucose, and acacia. 
   
   
       18 . The method of  claim 16 , wherein said carbohydrate is trehalose, sucrose or mixtures thereof. 
   
   
       19 . The method of  claim 1 , wherein said lyophilized cells are stored at a temperature from about room temperature to about −180° C. 
   
   
       20 . The method of  claim 1 , wherein said lyophilized cells are stored at about 4° C. to about −80° C. 
   
   
       21 . The method of  claim 1 , wherein said lyophilized cells are stored at about −20° C. to about −80° C. 
   
   
       22 . The method of  claim 1 , wherein said lyophilized cells are stored at about −20° C. 
   
   
       23 . The method of  claim 1 , wherein said lyophilized cells have a transformation efficiency of at least about 1×10 6  transformants per μg of DNA. 
   
   
       24 . The method of  claim 1 , wherein the transformation efficiency of said lyophilized cells is substantially retained after storage. 
   
   
       25 . The method of  claim 24 , wherein said storage is from 0 to about 450 days. 
   
   
       26 . The method of  claim 24 , wherein said storage is from about 240 days to about 365 days. 
   
   
       27 . The method of  claim 24 , wherein said storage is from about 365 days to about 450 days. 
   
   
       28 . Competent cells produced by the method of any one of  claim 1 ,  12 , or  15 . 
   
   
       29 . The competent cells of  claim 28 , wherein said competent cells are  Escherichia  cells. 
   
   
       30 . The competent cells of  claim 28 , wherein said cells are  E. coli  cells. 
   
   
       31 . A method for transforming a lyophilized competent cell comprising
 (a) obtaining a lyophilized competent cell produced by the method of any one of  claim 1 ,  12 , or  15 ;   (b) mixing said cell with a DNA molecule; and   (c) incubating said mixture under conditions sufficient to transform said cell with said DNA molecule.   
   
   
       32 . The method of  claim 31 , wherein said cells are  Escherichia  cells. 
   
   
       33 . The method of  claim 32 , wherein said cells are  E. coli  cells. 
   
   
       34 . The method of  claim 31 , wherein said DNA molecule is a vector. 
   
   
       35 . The method of  claim 31 , which further comprises rehydrating said lyophilized cells in a competence buffer prior to said mixing step. 
   
   
       36 . The method of  claim 31 , wherein said mixing step involves mixing said lyophilized cells with a competence buffer and the DNA molecule. 
   
   
       37 . Transformed competent cells produced by the method of  claim 31 . 
   
   
       38 . A method of producing a desired protein comprising
 (a) obtaining a lyophilized competent cell produced by the method of any one of  claim 1 ,  12  or  15 ;   (b) transforming said cell with a DNA molecule capable of expressing said desired protein; and   (c) culturing said transformed cell under conditions sufficient to produce said desired protein.   
   
   
       39 . The method of  claim 38 , wherein said cell is an  Escherichia  cell. 
   
   
       40 . The method of  claim 39 , wherein said cell is an  E. coli  cell. 
   
   
       41 . The method of  claim 38 , wherein said DNA molecule is a vector. 
   
   
       42 . The method of  claim 38 , wherein said transforming step comprises mixing said cell with competence buffer and said DNA molecule. 
   
   
       43 . A protein produced by the method of  claim 36 .