US4910140A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Electroporation of prokaryotic cells
Assignee: Bio Rad Laboratories Inc
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Filing: 1988-04-18
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Granted: 1990-03-20
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Expires: 2008-04-18
Abstract
Abstract Electroporation of prokaryotic cells is achieved by the application of high-intensity electric fields of short duration to reversibly permeabilize the cell walls. Preferably, the field strength of the applied will be at least about 5 kV/cm and the duration of the exposure will be in the range from 2 to 20 msec. Macromolecules may be introduced to the cells or obtained from the cells during this period of cell wall permeability. Most commonly, the method will be utilized for transformation of the cells with nucleic acids. Very high transformation efficiencies in the range from 10 9 to 10 10 cells/μg and transformation frequencies approaching 80% and above have been achieved.
Inventors
- 👤 William J. Dower
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 11:19
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/87; C12M; C12M35/00; C12M35/02; C12N13/00; C12N15/63; C12N15/64
Claims Analysis
Total Claims: 17
Claim 1
Independent Claim
124 words
A method for transforming bacterial cells, said method comprising: suspending the bacterial cells in a sample volume characterized by a low electrical conductivity solution of at least 1000Ω and a cell density of at least about 5×10 9 cells/ml while the walls of said cells remain substantially intact; subjecting the cells while in the solution and in the presence of nucleic acids at a concentration of at least about 1 pg/ml to an electric pulse across the sample volume, said pulse having a field strength of at least about 5 kV/cm for a time period sufficient to permeabilize the cell wall; and transferring the cells substantially immediately after being subjected to the electric pulse to a media capable of supporting growth of the cells.
Claim 2
Independent Claim
124 words
A method for transforming bacterial cells, said method comprising: suspending the bacterial cells in a sample volume characterized by a low electrical conductivity solution of at least 1000Ω and a cell density of at least about 5×10 9 cells/ml while the walls of said cells remain substantially intact; subjecting the cells while in the solution and in the presence of nucleic acids at a concentration of at least about 1 pg/ml to an electric pulse across the sample volume, said pulse having a field strength of at least about 5 kV/cm for a time period sufficient to permeabilize the cell wall; and transferring the cells substantially immediately after being subjected to the electric pulse to a media capable of supporting growth of the cells.
Claim 3
Dependent Claim
24 words
A method as in claim 1, wherein the solution has a salt concentration which results in an electrical resistance of at least about 1000Ω.
Claim 4
Dependent Claim
23 words
A method as in claim 1, wherein the time period of the pulse is in the range from about 2 to 20 msec.
Claim 5
Dependent Claim
28 words
A method as in claim 1, wherein the cell suspension is chilled to a temperature below about 10° C. prior to subjecting the cells to the electrical pulse.
Claim 6
Dependent Claim
23 words
A method as in claim 1, wherein the volume of the solution is in the range from about 1 μL to 1 ml.
Claim 7
Dependent Claim
12 words
A method as in claim 1, wherein the nucleic acids are circular.
Claim 8
Dependent Claim
12 words
A method as in claim 1, wherein the nucleic acids are linear.
Claim 9
Dependent Claim
14 words
A method as in claim 1, wherein the nucleic acids include a detectable marker.
Claim 10
Dependent Claim
15 words
A method as in claim 1, wherein the nucleic acids are free from detectable markers.
Claim 11
Dependent Claim
21 words
A method as in claim 1, wherein the nucleic acids are free from antibiotic resistance genes and heavy metal resistance genes.
Claim 12
Dependent Claim
24 words
A method as in claim 1, wherein the nucleic acids are present in the solution at a concentration of at least about 5 μg/ml.
Claim 13
Dependent Claim
20 words
A method as in claim 5, wherein the volume is in the range from about 10 μL to 100 μL.
Claim 14
Independent Claim
128 words
A method for transforming bacterial cells, said method comprising: suspensing the bacterial cells in a sample volume characterized by a low electrical conductivity solution of at least 1000Ω and a cell density of at least about 5×10 9 cells/ml, said cells not having been chemically treated to induce cell wall permeability; subjecting the cell while in the solution and in the presence of nucleic acids at a concentration of at least about 1 pg/ml to an electric pulse across the sample volume, said electric pulse having a field strength of at least about 5 kV/cm for a time period sufficient to permeabilize the cell wall; and transferring the cells substantially immediately after being subjected to the electrical pulse to a media capable of supporting growth of the cells.
Claim 15
Independent Claim
128 words
A method for transforming bacterial cells, said method comprising: suspensing the bacterial cells in a sample volume characterized by a low electrical conductivity solution of at least 1000Ω and a cell density of at least about 5×10 9 cells/ml, said cells not having been chemically treated to induce cell wall permeability; subjecting the cell while in the solution and in the presence of nucleic acids at a concentration of at least about 1 pg/ml to an electric pulse across the sample volume, said electric pulse having a field strength of at least about 5 kV/cm for a time period sufficient to permeabilize the cell wall; and transferring the cells substantially immediately after being subjected to the electrical pulse to a media capable of supporting growth of the cells.
Claim 16
Independent Claim
120 words
A method for transforming bacterial cells having a cell wall, said method comprising: suspending the bacterial cells in a sample volume characterized by a low electrical conductivity solution of at least 1000Ω and a cell density of at least about 5×10 9 cells/ml; subjecting the cells while in the solution and in the presence of nucleic acids at a concentration of at least about 1 pg/ml to an electric pulse across the sample volume, said electric pulse having a field strength of at least about 5 kV/cm for a time period sufficient to permeabilize the cell wall; and transferring the cells to a media capable of supporting growth of the cells within 60 seconds of exposure to the electric pulse.
Claim 17
Independent Claim
120 words
A method for transforming bacterial cells having a cell wall, said method comprising: suspending the bacterial cells in a sample volume characterized by a low electrical conductivity solution of at least 1000Ω and a cell density of at least about 5×10 9 cells/ml; subjecting the cells while in the solution and in the presence of nucleic acids at a concentration of at least about 1 pg/ml to an electric pulse across the sample volume, said electric pulse having a field strength of at least about 5 kV/cm for a time period sufficient to permeabilize the cell wall; and transferring the cells to a media capable of supporting growth of the cells within 60 seconds of exposure to the electric pulse.
References Cited (Backward Citations)
US4081340A
Cited
US4849355A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1993-03-19 | 1993-09-18 | 1994-03-20 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1997-03-19 | 1997-09-18 | 1998-03-20 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2001-03-19 | 2001-09-18 | 2002-03-20 | $7700.00 | Window Closed / Lapsed |