Room temperature stable competent cells
The invention provides storage-stable competent cells which retain good viability over long periods of time at room temperature (e.g., least one month). In one embodiment, a composition is provided comprising a mixture of competent cells and a glass-forming matrix material. Methods for generating room temperature stable competent cells are also provided.
1 . A method for generating storage-stable competent cells, which comprises drying competent cells at a temperature greater than freezing so as to generate storage-stable competent cells, wherein said cells are not freeze-dried.
2 . The method of claim 1 , wherein said cells are bacterial cells.
3 . The method of claim 2 , wherein said bacterial cells are gram negative cells.
4 . The method of claim 1 , wherein said cells are made competent by exposure to an electroporation buffer.
5 . The method of claim 1 , wherein the cells are dried for a single uniform temperature.
6 . The method of claim 1 or 5 , wherein the cells are dried for at least 8 hours.
7 . The method of claim 1 , wherein the cells are made competent by exposure to a chemical agent.
8 . The method of claim 7 , wherein said chemical agent is CaCl 2 .
9 . The method of claim 1 , wherein said competent cells can be stored at temperatures above −80° C. for at least one month and maintain transformation efficiencies of at least 10 5 transformants/μg DNA.
10 . The method of claim 9 , wherein said competent cells can be stored at temperatures of −20° C. or above for at least one month and maintain transformation efficiencies of at least 10 5 transformants/μg DNA.
11 . The method of claim 10 , wherein said competent cells can be stored at temperatures of 0° C. or above for at least one month and maintain transformation efficiencies of at least 10 5 transformants/μg DNA.
12 . The method of claim 11 , wherein said competent cells can be stored at temperatures of 4° C. or above for at least one month and maintain transformation efficiencies of at least 10 5 transformants/μg DNA.
13 . The method of claim 12 , wherein said competent cells can be stored at temperatures of 15° C. or above for at least one month and maintain transformation efficiencies of at least 10 5 transformants/μg DNA.
14 . The method of claim 11 , wherein said competent cells can be stored at temperatures of 20° C. or above for at least one month and maintain transformation efficiencies of at least 10 5 transformants/μg DNA.
15 . The method of claim 1 , wherein said cells are dried at a temperature above 0° C.
16 . The method of claim 15 , wherein said cells are dried at a temperature above 4° C.
17 . The method of claim 16 , wherein said cells are dried at a temperature at or above room temperature.
18 . The method of claim 17 , wherein said cells are dried at 30° C.
19 . The method of claim 1 , wherein said competent cells are exposed to non-atmosphere pressure during drying.
20 . The method of claim 1 , wherein said competent cells are dried under vacuum.
21 . The method of claim 1 , wherein said competent cells are dried in the presence of a glass-forming matrix material.
22 . The method of claim 21 , wherein said glass-forming matrix material comprises at least one carbohydrate.
23 . The method of claim 22 , wherein said at least one carbohydrate comprises a saccharide.
24 . The method of claim 21 , wherein the glass-forming matrix is water-soluble.
25 . The method of claim 23 , wherein said saccharide is selected from the group consisting of a disaccharide, an oligosaccharide, a polysaccharide, a sugar alcohol, a sugar ether, a sugar acid, derivatives thereof, and combinations thereof.
26 . The method of claim 23 , wherein said saccharide is a non-reducing sugar.
27 . The method of claim 23 , wherein said saccharide is selected from the group consisting of trehalose, sucrose, melzitose, raffinose, maltitol, sorbose, lactitol, dextrose, derivatives thereof, and combinations thereof.
28 . The method of claim 23 , wherein said saccharide is a polysaccharide is selected from the group consisting of amylose, FICOLL™, dextrin, starch, dextran, and polydextrose.
29 . The method of claim 21 , wherein said glass-forming matrix material comprises a polyol.
30 . The method of claim 29 , wherein said polyol is selected from the group consisting of a sugar polyol, propylene glycol, polyethylene glycol, derivatives thereof, and combinations thereof.
31 . The method according to claim 21 , wherein said glass-forming matrix material comprises a polymer selected from the group consisting of polyvinylpyrolidone, polyacrylamide, polyethyleneimine, and albumen.
32 . The method of claim 22 , wherein the concentration of said carbohydrate is at least 20% (weight/volume).
33 . The method according to claim 22 , wherein said carbohydrate comprises a saccharide and a sugar alcohol.
34 . The method according to claim 33 , wherein said saccharide is trehalose.
35 . The method according to claim 33 or 34 , wherein said sugar alcohol is sorbitol.
36 . The method according to claim 33 , wherein said saccharide is a hydrated saccharide.
37 . The method of claim 36 , further comprising the step of storing said competent cells at a temperature at or above −20° C.
38 . The method of claim 37 , further comprising the step of storing said competent cells at a temperature at or above 0° C.
39 . The method of claim 38 , further comprising the step of storing said competent cells at a temperature at or above 4° C.
40 . The method of claim 39 , further comprising the step of storing said competent cells at a temperature at or above 15° C.
41 . The method of claim 40 , further comprising the step of storing said competent cells at a temperature at or above room temperature.
42 . The method of claim 37 , wherein said competent cells are stored in a sealed pouch.
43 . A method of transforming cells with exogenous nucleic acids comprising, obtaining cells generated according to the method of claim 1 , rehydrating the cells, and contacting the cells with said nucleic acids.
44 . The method of claim 43 , further comprising the step of exposing the cells to at least one electrical pulse.
45 . The method of claim 43 , wherein said cells are rehydrated in transformation buffer or electroporation buffer.
46 . The method of claim 43 or 44 , wherein said cells exhibit transformation efficiencies of at least 1×10 5 transformants/μg DNA.
47 . A composition comprising a glass-forming matrix material and competent cells, wherein the glass transition temperature (Tg) of the matrix-cell mixture is greater than 15° C.
48 . The composition of claim 47 , wherein the glass transition temperature (Tg) of the matrix-cell mixture is greater than room temperature.
49 . The composition of claim 48 , wherein the glass transition temperature (Tg) of the matrix-cell mixture is greater than 20° C.
50 . The composition of claim 49 , wherein the glass transition temperature (Tg) of the matrix-cell mixture is greater than 25° C.
51 . The composition of claim 50 , wherein the glass transition temperature (Tg) of the matrix-cell mixture is greater than 30° C.
52 . The composition of claim 51 , wherein the glass transition temperature (Tg) of the matrix-cell mixture is greater than 40° C.
53 . The composition of claim 52 , wherein the glass transition temperature (Tg) of the matrix-cell mixture is greater than 45° C.
54 . The composition of claim 53 , wherein the glass transition temperature (Tg) of the matrix-cell mixture is greater than 50° C.
55 . The composition of claim 54 , wherein the glass transition temperature (Tg) of the matrix-cell mixture is greater than 60° C.
56 . The composition of claim 47 , wherein the transformation efficiency of said cells comprises at least 10 5 transformants/μg DNA.
57 . The composition of claim 47 , wherein the transformation efficiency of said cells comprises at least 10 6 transformants/μg DNA.
58 . The composition of claim 47 , wherein said glass-forming matrix comprises at least one carbohydrate.
59 . The composition of claim 58 , wherein said carbohydrate comprises a saccharide.
60 . The composition of claim 59 , wherein said saccharide is selected from the group consisting of a disaccharide, an oligosaccharide, a polysaccharide, a sugar alcohol, a sugar ether, a sugar acid, derivatives thereof, and combinations thereof.
61 . The composition of claim 59 , wherein said saccharide comprises a non-reducing sugar.
62 . The composition of claim 59 , wherein said saccharide is selected from the group consisting of trehalose, sucrose, melzitose, raffinose, maltitol, sorbose, lactitol, dextrose, derivatives thereof, and combinations thereof.
63 . The composition of claim 59 , wherein said saccharide comprises a polysaccharide selected from the group consisting of amylose, FICOLL™, dextrin, starch, dextran, and polydextrose.
64 . The composition of claim 58 , wherein said carbohydrate comprises a saccharide and a sugar alcohol.
65 . The composition of claim 64 , wherein said saccharide comprises trehalose.
66 . The composition of claim 64 , wherein said sugar alcohol comprises sorbitol.
67 . The composition of claim 47 , wherein said glass-forming matrix material comprises a polyol.
68 . The composition of claim 67 , wherein said polyol is selected from the group consisting of a sugar polyol, propylene glycol, polyethylene glycol, derivatives thereof, and combinations thereof.
69 . The method according to claim 47 , wherein said glass-forming matrix material is a polymer selected from the group consisting of polyvinylpyrolidone, polyacrylamide, polyethyleneimine, and albumen.
70 . The composition of claim 47 , wherein at least 5% of said cells are viable upon rehydration.
71 . The composition of claim 70 , wherein at least 10% of said cells are viable upon rehydration.
72 . The composition of claim 71 , wherein at least 15% of said cells are viable upon rehydration.
73 . The composition of claim 72 , wherein at least 20% of said cells are viable upon rehydration.
74 . The composition of claim 71 , wherein at least 30% of said cells are viable upon rehydration.
75 . A kit comprising a composition according to claim 47 , wherein said matrix-cell mixture is stored in a sealed pouch.
76 . The kit of claim 75 , wherein the kit further comprises a sample of nucleic acids in a container which is separated from said sealed pouch.
77 . The kit according to claim 75 , wherein said nucleic acids are lyophilized.
78 . A method of producing a recombinant polypeptide comprising:
obtaining cells generated according to the method of claim 1;
rehydrating the cells;
contacting the cells with a nucleic acid encoding said recombinant polypeptide; and
growing said cells in a cell growth media under conditions in which the cells produce said polypeptide.
79 . The method of claim 78 , in which cells which have taken up said nucleic acid are separated from cells which have not taken up said nucleic acids.
80 . The method of claim 78 , wherein said recombinant polypeptide is isolated from said cells.