INDIRECT SONICATION FOR CELL EXTRACT PREPARATIONS FOR CELL-FREE PROTEIN SYNTHESIS
Methods of lysing cells, e.g., for use in methods of cell-free protein synthesis, are disclosed that include placing one or more vessels within a liquid in a reservoir, wherein each vessel contains one or more cells in a cell suspension; and applying sonic energy to the liquid within the reservoir during a plurality of predetermined time intervals and under conditions sufficient to disrupt cell walls and/or cell membranes of cells in the cell suspension, and to release into the cell suspension cell contents that include active transcription and/or translation machinery.
1 . A method of lysing cells, the method comprising:
placing one or more vessels within a liquid in a reservoir, wherein each vessel contains one or more cells in a cell suspension; and
applying sonic energy to the liquid within the reservoir during a plurality of predetermined time intervals and under conditions sufficient to disrupt cell walls and/or cell membranes of cells in the cell suspension, and to release into the cell suspension cell contents that include active transcription and/or translation machinery.
2 . The method of claim 1 , wherein the sonic energy is applied indirectly to the cell suspension under conditions sufficient to maintain the functionality of the transcription and translation machinery contained within the cells.
3 . The method of claim 1 or claim 2 , wherein the sonic energy is applied indirectly to the cell suspension for a total time period of about 30 seconds to about 6 hours, e.g., about 30, 40, 50, 60, 70, 80, 90, 100, or 120 seconds, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or 60 minutes, or about 1, 2, 3, 4, 5, or 6 hours.
4 . The method of claim 3 , wherein the predetermined time intervals are each about 5 to about 120 seconds, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, or 120 seconds, separated by gaps of about 5 to about 30 seconds, e.g., about 5, 10, 15, 20, 25, or 30 seconds, during which the sonic energy is not applied or is reduced.
5 . The method of claim 4 , wherein the predetermined time intervals are each about 5 seconds to about 20 seconds and the gaps between predetermined time intervals are about 10 to about 20 seconds, during which the sonic energy is not applied.
6 . The method of any one of claims 1 to 5 , wherein the total time period during which the sonic energy is applied is greater than or equal to 10 minutes and less than or equal to 20, 30, 40, 50 or 60 minutes.
7 . The method of any one of claims 1 to 6 , wherein the sonic energy is equal to an energy delivered by a sonicator with an amplitude set at about 50%.
8 . The method of any one of claims 1 to 7 , wherein a power density (W/mL reservoir) of the sonic energy is equal to the power density delivered by a sonicator with an amplitude set at about 50%.
9 . The method of any one of claims 1 to 8 , wherein an energy density (J/mL reservoir) of the sonic energy is equal to an energy density delivered by a sonicator with an amplitude set at about 50%.
10 . The method of any one of claims 1 to 9 , wherein a power density of the sonic energy is within a range of about 2 to about 3 W/mL of a volume of the reservoir.
11 . The method of any one of claims 1 to 10 , wherein an energy density of the sonic energy is within a range of about 2000 to about 5000 J/mL of a volume of the reservoir.
12 . The method of any one of claims 1 to 11 , further comprising maintaining a temperature of the liquid within the reservoir below a threshold temperature.
13 . The method of claim 12 , wherein the threshold temperature is at or below about 50° C., 40° C., 35° C., 30° C., 25° C., 20° C., 15° C., 10° C., or 5° C.
14 . The method of claim 12 or claim 13 , wherein the temperature of the liquid within the reservoir is maintained with a cooling system.
15 . The method of any one of claims 12 to 14 , wherein the temperature of the liquid within the reservoir is maintained by controlling a length of the predetermined time intervals and/or a length of the gaps between the predetermined time intervals.
16 . The method of any one of claims 1 to 15 , further comprising transmitting the sonic energy with sonic horn.
17 . The method of any one of claims 1 to 16 , wherein the vessels are part of a sample tray or plate.
18 . The method of claim 17 , wherein the sample tray or plate comprises a multi-well plate.
19 . The method of claim 18 , wherein the multi-well plate comprises 6, 24, 48, 96, 384, or 1536 wells.
20 . The method of any one of claims 1 to 19 , wherein the cells comprise bacterial cells, yeast cells, mammalian cells, insect cells, plant cells, and/or worm cells.
21 . The method of any one of claims 1 to 20 , wherein a frequency of the sonic energy is in a range of about 15 and about 30 kHz.
22 . The method of any one of claims 1 to 21 , wherein volumes of the cell suspensions are in a range between about 20 microliters and about 50 milliliters.
23 . The method of any one of claims 1 to 22 , wherein the liquid within the reservoir comprises one or more of water, one or more gels, and/or one or more organic solvents.
24 . The method of any one of claims 1 to 23 , wherein the vessels each have a volume of less than 2 milliliters.
25 . A method of cell-free protein synthesis, the method comprising:
placing one or more vessels within a liquid in a reservoir, wherein each vessel contains one or more cells in a cell suspension;
applying sonic energy to the liquid within the reservoir during a plurality of predetermined time intervals and under conditions sufficient to disrupt cell walls and/or cell membranes of cells in the cell suspension, and to release into the cell suspension cell contents that include active transcription and/or translation machinery;
harvesting and/or processing at least a portion of the cell contents; and
synthesizing proteins using the active transcription and/or translation machinery.
26 . The method of claim 25 , wherein the active transcription and/or translation machinery comprises one or more of ribosomes, tRNAs, synthetases, transcription factors, translation initiation factors, elongation factors, or release factors.
27 . The method of claim 25 , wherein the active transcription and/or translation machinery comprises ribosomes, tRNAs, synthetases, transcription factors, translation initiation factors, elongation factors, and release factors.
28 . The method of any one of claims 25 to 27 , further comprising maintaining a temperature of the liquid within the reservoir below a threshold temperature at or below about 50° C., 40° C., 35° C., 30° C., 25° C., 20° C., 15° C., 10° C., or 5° C.
29 . The method of any one of claims 25 to 28 , wherein synthesizing the proteins comprises combining the portion of the cell contents with one or more nucleic acid templates that encode one or more proteins.
30 . The method of any one of claims 25 to 29 , wherein the sonic energy is applied indirectly to the cell suspension under conditions sufficient to maintain the functionality of the transcription and translation machinery contained within the cells.
31 . The method of any one of claims 25 to 30 , wherein the sonic energy is applied indirectly to the cell suspension for a total time period of about 30 seconds to about 6 hours, e.g., about 30, 40, 50, 60, 70, 80, 90, 100, or 120 seconds, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or 60 minutes, or about 1, 2, 3, 4, 5, or 6 hours.
32 . The method of claim 31 , wherein the predetermined time intervals are each about 5 to about 120 seconds, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, or 120 seconds, separated by gaps of about 5 to about 30 seconds, e.g., about 5, 10, 15, 20, 25, or 30 seconds, during which the sonic energy is not applied or is reduced.
33 . The method of claim 32 , wherein the predetermined time intervals are each about 5 seconds to about 20 seconds and the gaps between predetermined time intervals are about 10 to about 20 seconds, during which the sonic energy is not applied.
34 . The method of any one of claims 25 to 33 , wherein the total time period during which the sonic energy is applied is greater than or equal to 10 minutes and less than or equal to 20, 30, 40, 50 or 60 minutes.
35 . The method of any one of claims 25 to 34 , wherein the sonic energy is equal to an energy delivered by a sonicator with an amplitude set at about 50%.
36 . The method of any one of claims 25 to 35 , wherein a power density (W/mL reservoir) of the sonic energy is equal to the power density delivered by a sonicator with an amplitude set at about 50%.
37 . The method of any one of claims 25 to 36 , wherein an energy density (J/mL reservoir) of the sonic energy is equal to an energy density delivered by a sonicator with an amplitude set at about 50%.
38 . The method of any one of claims 25 to 38 , wherein a power density of the sonic energy is within a range of about 2 to about 3 W/mL of a volume of the reservoir.
39 . The method of any one of claims 25 to 38 , wherein an energy density of the sonic energy is within a range of about 2000 to about 5000 J/mL of a volume of the reservoir.
40 . The method of any one of claims 28 to 39 , wherein the temperature of the liquid within the reservoir is maintained with a cooling system.
41 . The method of any one of claims 28 to 39 , wherein the temperature of the liquid within the reservoir is maintained by controlling a length of the predetermined time intervals and/or a length of the gaps between the predetermined time intervals.
42 . The method of any one of claims 25 to 41 , further comprising transmitting the sonic energy with sonic horn.
43 . The method of any one of claims 25 to 42 , wherein the vessels are part of a sample tray or plate, e.g., a multi-well plate.
44 . The method of any one of claims 25 to 43 , wherein the cells comprise bacterial cells, yeast cells, mammalian cells, insect cells, plant cells, and/or worm cells.
45 . The method of any one of claims 25 to 44 , wherein a frequency of the sonic energy is in a range of about 15 and about 30 kHz.
46 . The method of any one of claims 25 to 45 , wherein volumes of the cell suspensions are in a range between about 20 microliters and about 50 milliliters.
47 . The method of any one of claims 25 to 46 , wherein the liquid within the reservoir comprises one or more of water, one or more gels, and/or one or more organic solvents.
48 . The method of any one of claims 25 to 47 , wherein the vessels each have a volume of less than 2 milliliters.