Isotachophoretic nucleic acid transformation of cells

Field of invention

The invention relates to a method for transforming cells with nucleic acid molecules.

Background

Transformation, the introduction of nucleic acid molecules, typically deoxyribonucleic acid (DNA) molecules, into bacteria, is one of the most common procedures in molecular biology. Transformation relies on the easy availability and fast growth rate of bacteria, which makes them ideal for storing and replicating recombinant plasmid DNA including those designed for expression in mammalian cells. Transformation success can be evaluated in two ways: efficiency of DNA uptake (transformation efficiency) and the proportion of cells that were transformed (transformation rate).

Chemical transformation (CT) and electroporation (EP) are the most commonly employed transformation methods. However, both approaches require a substantial number of cells per reaction (10 ⁹ -10 ¹¹ ). Further, CT and EP are tedious and requires time-consuming procedures to induce cell competence (over 4 hours) and in-house prepared competent cells may provide low transformation success. To overcome this limitation, high efficiency competent cells“ultra-“or“super-“competent cells are usually purchased. EP requires specialized pulse generators and a high cell death of about 50 to 60% for maximum transformation efficiency.

There is a continuing need to develop alternative methods for transforming cells with nucleic acid molecules. There is also a continuing need to develop techniques to transform non- competent cells. Advantageously, new methods may be able to transform cells with nucleic acid molecules with high rates of transformation, potentially enabling high-throughput transformations.

Summary

In one aspect, the invention provides a method for transforming a cell with a nucleic acid molecule, comprising applying a voltage to a solution comprising (i) the cell; (ii) the nucleic acid molecule; (iii) a leading electrolyte; and (iv) a terminating electrolyte; wherein when the nucleic acid molecule is single-stranded, the nucleic acid molecule comprises at least 25 bases. In another aspect, the invention provides a cell transformed by the method of the invention.

In a further aspect, the invention provides a kit comprising in separate parts (a) a leading electrolyte and (b) a terminating electrolyte; and at least one of (c) a nucleic acid molecule and/or (d) a cell.

In yet another aspect, the invention provides a kit comprising in separate parts (a) a leading electrolyte and (b) a terminating electrolyte; and wherein part (a) and/or part (b) further comprises at least one of (i) a nucleic acid molecule and/or (ii) a cell.

In a further aspect, there is provided the use of a leading electrolyte solution and a terminating electrolyte solution as reagents for performing the transformation of a cell with a nucleic acid molecule.

The present application also provides for the use of isotachophoresis to transform a cell with a nucleic acid molecule. In such uses, it is preferred that when the nucleic acid molecule is single-stranded, the nucleic acid molecule comprises at least 25 bases.

In yet another aspect, the invention provides a capillary electrophoresis apparatus configured for performing the method as described above.

Brief Description of Drawings

The present invention will be further described, by way of example only, with reference to the accompanying drawings, in which:

Figure 1a shows schematics of the Isotachophoresis (ITP)-based transformation method described in Example 1 : (1 ) the capillary is filled with leading electrolyte (LE); (2) DNA in LE and cells in terminating electrolyte (TE) are hydrodynamically injected (9.0 psi for 10 and 5 seconds) into the capillary; (3) the inlet vial is switched to TE and (i) -16 kV is applied, and cells and plasmid in the ITP band are detected/collected; or (ii) a voltage of -16 kV and 1 .3 psi counter-pressure are applied, the counter-pressure is removed after the desired period to allow the ITP band to move and the cells and plasmid to be detected/collected.

Figure 1 b shows overlapping elution peaks confirming detection of plasmids and cells at the same elution time validating the ITP method.

Figure 1 c shows images of transformed colonies obtained (1 ) without counter pressure or (2) with counter-pressure for 21 minutes.

Figure 2 shows a chart of viability (left axis), transformation rate (left axis) and

transformation efficiency (right axis) as a function of the counter-pressure time.

Figures 3A and B show charts of (A) transformation efficiency and (B) transformation rate for transformations by CT and EP using 1 -2 × 10 ¹⁰ and 1 -2 × 10 ⁵ competent and non- competent cells and the ITP-based transformation of the invention.

Figure 4 shows a chart of transformation rate (left axis) and transformation efficiency (right axis) as a function of plasmid size obtained by the procedure described in Example 1 .

Figures 5A and B show electropherograms of (A) pUC18 cells suspended in TE containing 1 % DMSO, stained with Midori Green Advance DNA stain; and (B) E. coli TOP 10 cells suspended in TE containing 1 % DMSO, stained with SYTO59.

Figure 6 shows a chart of Transformation efficiency obtained by ITP-based transformation as a function of cells suspended in TE and TE containing 1 % (v/v) DMSO.

Figure 7 shows a chart of TOP10 cell viability as a function of DMSO concentration (% v/v) in the terminating electrolyte during the ITP method of Example 1.

Figure 8 shows electropherograms of TOP10 cells at 2-3 x 10 ² cells stained with mM SYTO9 suspended in TE with 0.1%, 1 % and 10% (v/v) of DMSO.

Figure 9 shows a current profile of human Jurkat T cells hydrodynamically injected (9 psi, 15 seconds) exposed to a voltage varying from 1 to 10 kV for 30 seconds followed by pressure (50 psi) for 2 minutes.

Figure 10 shows the viability of Jurkat T cells in medium, phosphate buffered saline (PBS), sodium phosphate and sodium HEPES over incubation time at ambient temperature.

Figure 11 shows an electropherogram of fluorescent carboxyl-functionalised microspheres with mean diameter from 0.51 mm and 15.45 mm. Experimental conditions: 40 cm × 100 mm ID Rtx Wax GC Column (fused silica) with detection window at 30 cm. At each analysis, the capillary was filled with LE 0.16 M sodium-phosphate (pH 7.3) followed by the injection of cells in TE 0.16 M sodium-HEPES (pH 7.3) at field strength of 0.075 kV/cm (3 kV) at reversed polarity.

Figure 12 shows a schematic of the ITP-based transformation method. (A1 ) Capillary is filled with LE. (A2) DNA in LE and cells in TE are hydrodynamically injected (5.0 psi for 5 and 10 seconds). (A3) The inlet vial is switched to TE and (i) -3.0 kV is applied, and cells and plasmid in the ITP band are detected/collected. (B) Validation of the ITP method showing the focusing of plasmid and cells within the same peak at the ITP interface (C). Protein (GFP) expression is assayed 48 hours after ITP transfection by fluorescence microscopy (C1 ) and phase contrast (C2).

Figure 13 shows microscopy images of: 3-4×10 ⁶ Jurkat T cells transformed with pCMV6- AC-GFP by EP (fluorescence 1 and phase contrast 2), 1-2×10 ⁴ Jurkat T cells transformed with pCMV6-AC-GFP by EP (fluorescence 3 and phase contrast 4), 1-2×10 ⁴ Jurkat T cells transformed with pCMV6-AC-GFP by ITP (fluorescence 5 and phase contrast 6), and 1 - 2×10 ⁴ Jurkat T cells transformed with pCMV6 by ITP (fluorescence 7 and phase contrast 8). Figure 14 shows transformation success obtained via EP using 3-4 × 10 ⁶ and 1 -2 × 10 ⁴ Jurkat T cells and their comparison with the ITP-based transfection. (A) Transfection efficiency. (B) Transfection rate.

Description of embodiment(s)

Before describing the present invention in detail, it is to be understood that this invention is not limited to particularly exemplified embodiments, such as the cells or methods of preparing the cells, which may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting.

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. However, publications mentioned herein are cited for the purpose of describing and disclosing the protocols and reagents which are reported in the publications and which might be used in connection with the invention.

Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

The term“transformation” and variations thereof, such as“transforming” and“transforms”, relate to the method of introducing a nucleic acid molecule into a cell, such as a prokaryotic or eukaryotic cell or a subcellular organelle such as a mitochondria or a plastid such as a chloroplast. In some embodiments, the cell is a eukaryotic cell specifically.

The term“electrolyte”, unless the context requires otherwise, refers to a solution comprising anions and cations dissolved in water. Under an electrical potential, the anions move to the electrode with lack of electrons (anode), while the cations move to the electrode with abundance of electrons (cathode). It is noted that the electrodes may be cathodes, anodes or grounded electrodes.

It must be noted that as used herein and in the appended claims, the singular forms“a,”

“an,” and“the” include plural reference unless the context clearly dictates otherwise.

Thus, for example, a reference to“a cell” may include one or more cells, and so forth.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any materials and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred materials and methods are now described.

The term“(s)” following a noun contemplates the singular or plural form, or both.

The term“and/or” can mean“and” or“or”.

Unless the context requires otherwise, all percentages referred to herein are percentages by weight of the composition.

Unless the context requires otherwise, all amounts referred to herein are intended to be amounts by weight.

Various features of the invention are described with reference to a certain value, or range of values. These values are intended to relate to the results of the various appropriate measurement techniques, and therefore should be interpreted as including a margin of error inherent in any particular measurement technique. Some of the values referred to herein are denoted by the term“about” to at least in part account for this variability. The term“about”, when used to describe a value, may mean an amount within ±25%, ±10%, ±5%, ±1 % or ±0.1 % of that value.

The term“comprising” is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention. When interpreting statements in this specification that include that term, the features, prefaced by that term in each statement, all need to be present but other features can also be present. Related terms such as“comprise” and“comprised” are to be interpreted in the same manner.

Transformation methods

The invention relates to a method for transforming a cell with a nucleic acid molecule. The method comprises applying a voltage to a solution comprising (i) the cell; (ii) the nucleic acid molecule; (iii) a leading electrolyte; and (iv) a terminating electrolyte. When the nucleic acid molecule is single-stranded, the nucleic acid molecule comprises at least 25 bases.

The inventors have surprisingly shown that cells can be transformed with certain nucleic acid molecules by subjecting the cells and nucleic acid molecules to isotachophoretic conditions. The transformation rate is higher than common transformation protocols, without sacrificing transformation efficiency. The rapid method times - both in terms of omission of the cell competency requirement and high rate of transformation - may enable rapid technologies relying on rapid cellular transformation(s), such as high-throughput transformation.

Following from the above, the method may further comprise:

- Applying the voltage to the solution, without performing steps to induce cell

competence.

The method may further comprise:

- Introducing a leading electrolyte into a vessel;

- Introducing the terminating electrolyte into the vessel subsequent to the introduction of the leading electrolyte into the vessel,

- Introducing the cell into the vessel in combination with one of the lead electrolyte and the terminating electrolyte, or separately;

- Introducing the nucleic acid molecule into the vessel in combination with one of the terminating electrolyte and the leading electrolyte, or separately; and

- forming said solution from the lead electrolyte, cell, nucleic acid molecule and

terminating electrolyte in the vessel prior to application of the voltage to the solution.

In some embodiments, the above method further comprises:

- completing the steps of introducing the leading electrolyte, terminating electrolyte, cell and nucleic acid molecule into the vessel for an array of vessels; and

- applying the voltage to the solutions in the vessels in the array of vessels to

transform the cells in each vessel in the array with the nucleic acid molecule in each vessel in the array.

In the method of the present application, the transformation may be effected within a period of not more than 10 minutes, such as not more than 9 minutes, not more than 8 minutes, not more than 7 minutes or not more than 6 minutes. The time period may be between 2 minutes and 6 minutes. This provides the ability to perform a high throughput of

transformations. This is particularly the case when performed using an array of vessels.

The number of vessels in the array may be at least 16, 24, 32, 40, 48, 56, 64, 72, 80, 88, 96 or higher.

Isotachophoresis (ITP) is a variant of electrophoresis. ITP is a method that separates charged molecules based on their ionic mobility in an electric field, as for all electrophoretic techniques. The charged molecules intended to be separated or concentrated through ITP are sometimes referred to as analytes. In the context of the present application, the cell and/or the nucleic acid molecules may be viewed as the analyte or analytes. ITP also includes in the analyte solution, ions that have an ionic mobility faster than the analyte (leading electrolyte - LE) and ions that have an ionic mobility slower than the analyte (terminating electrolyte - TE). Thus, during ITP, exposing a solution comprising an analyte, an LE and a TE to an electric field, causes the formation of three zones within the solution. The first zone comprises the LE, the second zone comprises the analyte and the third zone comprises the TE. The location of the first and third zone will be dictated by the charge of the LE and TE and the direction of the electric field. The second zone is sandwiched between the first and second zones and has a length and concentration that is dependent on the initial concentration of the analyte and the concentration of the leading ion. If there are more than one analyte with a mobility between the leading and terminating ions, then they will both migrate in the second zone, until the concentration of one of these analytes reaches its steady state concentration.

The inventors discovered that establishing ITP in a solution comprising cells and nucleic acid molecules, creates a zone of high concentration of both the cells and the nucleic acid molecules (see Figure 1 A). The increased concentration of both cells and nucleic acid molecules helps drive the transformation method. Further, it is believed that as the cells are subjected to the electric field that some electroporation also occurs; however, the method of the invention is superior to transformation by traditional electroporation as it is capable of transforming non-competent cells. Also, as the cells are concentrated during ITP, there is less processing of the cells to increase the concentration prior to ITP. In the method of the invention, the cells and nucleic acid molecules are treated as the analytes in separatory ITP. The ITP thus focusses the cells and nucleic acid molecules into a concentrated zone of the solution sandwiched between the LE and the TE. This method therefore greatly accelerates the rate of transformation allowing comparable transformation efficiencies with EP and CT techniques in a fraction of the time.

The transformation rate may be calculated by dividing the number of transformed Colony- Forming Units (CFU) by the injected CFU multiplied by 100. Colony count may be determined by counting on incubated plates. As shown in Figure 3B, transformation rates for competent and non-competent cells using EP or CT are less than 0.05%. Accordingly, the minimum transformation rate for the method of the invention may be typically at least about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1 %, about 0.15%, about 0.2%, about 0.25% or about 0.3%. In some embodiments, the maximum

transformation efficiency may be not more than about 1 %, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.45%, about 0.4%, or about 0.35%. The

transformation rate may be between any of these minimum and maximum rates, such as about 0.1 % to about 1 %, about 0.1 % to about 0.5%, about 0.1 % to about 0.4% or about 0.25% to about 0.35%.

The transformation efficiency may be calculated by dividing the number of transformed colony-forming units (CFU) by the amount of nucleic acid molecule present in the ITP solution (pg). In some embodiments, the minimum transformation efficiency achieved may be at least about 1 CFU/mg nucleic acid molecule, about 50CFU/mg nucleic acid molecule, about 100CFU/mg nucleic acid molecule, about 500CFU/mg nucleic acid molecule, about 1 ,000CFU/mg nucleic acid molecule, about 2,000CFU/mg nucleic acid molecule, about 3,000CFU/mg nucleic acid molecule, about 4,000CFU/mg nucleic acid molecule, about 5,000CFU/mg nucleic acid molecule, about 6000CFU/mg nucleic acid molecule, about 7000CFU/mg nucleic acid molecule, about 8000CFU/mg nucleic acid molecule, or about 8500CFU/mg nucleic acid molecule. The maximum transformation efficiency achieved may be not more than about 1 ,000,000CFU/mg nucleic acid molecule, about 800,000CFU/mg nucleic acid molecule, about 700,000CFU/mg nucleic acid molecule, about 600,000CFU/mg nucleic acid molecule, about 500,000CFU/mg nucleic acid molecule, about 400,000CFU/mg nucleic acid molecule, about 300,000CFU/mg nucleic acid molecule, about 200,000CFU/mg nucleic acid molecule, about 100,000CFU/mg nucleic acid molecule, about 50,000CFU/mg nucleic acid molecule, about 10,000CFU/mg nucleic acid molecule, or about 9,000CFU/mg nucleic acid molecule. The transformation efficiency may be between any of these minimum and maximum rates, such as from about 1 CFU/mg nucleic acid molecule to about

1 ,000,000CFU/mg nucleic acid molecule or about 5,000CFU/mg nucleic acid molecule to about 10,000CFU/mg nucleic acid molecule.

The transformation method of the invention comprises applying a voltage to the solution.

The voltage may be applied by any suitable means provided that a sufficient electric field is created and/or maintained within the solution to establish isotachophoresis and focus the cells and nucleic acid molecules within the same zone of the solution. Therefore, in some embodiments, the method comprises creating an electric field in the solution. Since the application of the voltage through the solution creates the electric field, the step of creating the electric field and applying the voltage may be used interchangeably.

In some embodiments, the voltage is applied to create a minimum electric field strength at least about 100 V/cm, about 120 V/cm, about 150 V/cm, about 180 V/cm, about 200 V/cm, about 220 V/cm, about 240 V/cm, about 250 V/cm, about 300 V/cm, about 350 V/cm, about 400 V/cm, about 450 V/cm, about 500 V/cm, about 600 V/cm, about 700 V/cm, about 800 V/cm, about 900 V/cm, about 1000 V/cm or greater. The voltage may be applied to create a maximum electric field strength of not more than about 2000 V/cm, about

1900 V/cm, about 1800 V/cm, about 1700 V/cm, about 1600 V/cm, about 1500 V/cm, about 1400 V/cm, about 1300 V/cm, about 1200 V/cm, about 1 100 V/cm, about 1000 V/cm or less. The electric field strength may be between any of these minimum and maximum amounts. In some embodiments, the electric field strength may be from about 100 V/cm to about 2000 V/cm, about 100 V/cm to about 1200 V/cm or about 700 V/cm to about 1800 V/cm.

The cell survival rate may be at least about 40%, about 50%, about 55% or about 60%.

In some embodiments, to establish the above electric fields, the magnitude of the applied voltage may be not more than about 30kV, such as not more than about 20kV, about 16kV, about 15kV, about 10kV or about 5kV. Typically, the minimum voltage applied to the solution may be at least about 0.1 V, about 0.5V or about 1 V. In some embodiments, the voltage is between any of the above minimum voltages to the maximum voltages, such as from about 0.1 V to about 30kV, about 0.1 V to about 16kV or about 1 V to about 5kV.

Typically, the electric field is created within the solution by applying a voltage across two electrodes. The electric field parameters will depend in part on voltage source, magnitude and polarity of the electric field, resistance of the solution, nature and positioning of the electrodes amongst other factors. Thus, altering any of these variables may impact on the isotachophoresis established within the electric field.

The method of the invention may be carried out in any suitable vessel (or container). The vessel may be open or closed. Examples of suitable vessels for the solution include a capillary, a microchannel, a column, a reservoir, or a syringe. The vessel comprises means to apply the voltage through the solution. Typically, the vessel comprises two electrodes positioned apart by a defined distance. The distance between the electrodes may be used to calculate the electric field created in the solution.

The vessel may comprise a matrix. In some embodiments, the matrix extends the entire distance between the two electrodes, whereas in others the matrix may extend only part of the distance between the electrodes or may extend beyond one or both electrodes. In some embodiments the matrix is a gel. Any suitable gel may be used including agarose gels, acrylamide gels and silica gels. Inclusion of a matrix in the vessel may assist separate components in the solution and may be used to separate components after cellular transformation (product purification) or used prior to transformation (purification of reagents).

In some embodiments, the voltage is applied for a maximum time of not more than about 120 minutes (m), such as not more than about 100m, 90m, 60m, 55m, 50m, 45m, 40m,

35m, 30m, 25m, 20m, 15m, 10m, 5m or 3m. The voltage may be applied for a minimum time of at least about 0.1 milliseconds (ms), such as at least about 0.5ms, 1 ms, 0.5 seconds (s),

1 s, 30s, 1 m, 2m, 3m, 5m, 10m or 15m. The voltage may be applied for any time between any of the minimum times to any of the maximum times mentioned above, such as from about 0.1 ms to about 120m, about 0.1 ms to about 30m or about 5m to about 30m.

In some embodiments, the method further comprises applying the voltage to the solution under increased pressure. The increased pressure may be a pressure above atmospheric pressure. The pressure applied to the solution is preferably a counter pressure. By“counter pressure” it is meant that pressure is applied in the opposite direction to the direction of migration of the cells and/or nucleic acid molecules in the solution undergoing ITP. The counter pressure may be applied by any suitable means known in the art. The counter pressure may have a maximum magnitude of about 2psi, such as about 1.9, 1 .8, 1 .7, 1.6,

1 .5, 1.4 or 1.3 psi. The minimum counter pressure may be at least about 1 .01 psi, such as about 1 .1 , 1.15 or 1.2 psi. The counter pressure may be between any of these minimum and maximum pressures, such as from about 1.01 psi to about 2psi or 1.01 psi to about 1.5psi. Typically, the counter pressure is applied during the application of voltage to the solution. In some embodiments the counter pressure is applied for the entire time that the voltage is applied. The counter pressure, however, may be applied for only a portion of the time that the voltage is applied, or may be applied before and/or after voltage application. In some embodiments, the counter pressure is applied for a maximum time of not more than about 120m, such as not more than about 100m, 90m, 60m, 55m, 50m, 45m, 40m, 35m, 30m,

25m or 21 m. The counter pressure may be applied for a minimum time of at least about 0.1 ms, such as at least about 0.5ms, 1 ms, 0.5s, 1 s, 30s, 1 m, 2m, 3m, 5m, 10m or 15m. The voltage may be applied for any time between any of the minimum times to any of the maximum times mentioned above, such as from about 0.1 ms to about 120m, about 0.1 ms to about 30m or about 5m to about 30m. The application of counter pressure enables the period of time the cells are exposed (in contact) to (with) the nucleic acid molecules to be controlled independently of the electric field strength to effectively provide an incubation period. In addition, the counter pressure may assist to further increase the concentration of cells and nucleic acid molecules in the solution under ITP by narrowing the zone between the LE and TE. It should be further noted that a pressure may be applied by positive application of a pressure, or conversely, by application of a vacuum in an opposite direction. Usually the pressure is applied by means of a positive application of a pressure. In the case of a vessel having two ends, the pressure is applied to one end of the vessel counter to the direction of migration of cells and/or nucleic acid molecules.

In relation to the application of a counter pressure, it is noted that the pressure application impacts on the time taken for the migration of the cells and/or nucleic acid molecules in the solution undergoing ITP. By way of example, if the migration of cells and/or nucleic acid takes 5 minutes in the absence of a counter-pressure, a counter-pressure can be applied to slow the rate of migration. The rate of migration could be slowed by, for example, a factor of 2 or more (doubling the time in the example of 5 minutes to 10 minutes), or by a factor of 3 or more. As demonstrated in the examples, this application of counter-pressure assists with achieving higher transformation efficiency. While such counter-pressure application slows migration, the overall impact is a higher transformation efficiency and improved

transformation outcome.

In some embodiments, the method further comprises a step of conditioning the vessel. The conditioning step may comprise washing the vessel with one or more treating solutions and/or applying a voltage to the vessel. A suitable treating solution may be selected from one or more of a basic solution (e.g. 1 M sodium hydroxide), an acid solution (e.g. 1 M hydrochloric acid), the LE, the TE and combinations thereof. Typically, between each wash with a treating solution the vessel is also washed with the medium. In embodiments where the conditioning step comprises applying a voltage to the vessel, the vessel will preferably contain an electrolyte solution, such as LE or TE, preferably LE. The voltage is then applied across the electrolyte solution contained in the vessel, through applying a voltage potential across the pair of electrodes.

The method may also comprise one or more of the following steps:

- detecting the cells and/or nucleic acids,

- culturing the cells,

- isolating the transformed cells and/or

- expressing, purifying and overproducing a recombinant protein encoded by the

transformed cells.

Each of these additional steps may be carried out by techniques known in the art.

Solution

The method comprises applying a voltage to a solution. The solution comprises (i) a cell; (ii) a nucleic acid molecule; (iii) a leading electrolyte (LE); and (iv) a terminating electrolyte (TE). While each of components (i)-(iv) may be introduced into the vessel separately,“the solution” refers to the combination of these components through which the voltage is applied. For ease of reference, sometimes this solution is referred to herein as“the ITP solution”.

The ITP solution comprises a medium which solvates the ionic species of the LE, TE and nucleic acid molecule and disperses the cell. Any medium stable under ITP conditions and non-toxic to the cells prior to and following transformation may be used. Typically, the medium is a liquid under the transformation conditions and may comprise one or more solvents. Suitable solvents include water, acetonitrile, dimethylformamide (DMSO), and combinations thereof. The medium will preferably be the same as the solvent of the LE and TE or at least miscible with the solvent of the LE and TE. Typically the medium is filtered through 0.2mm sterile filters (Milex-GS Syringe Filter, Merck Millipore, Bayswater, Australia) and comprises 18.2 MW cm ^-1 Type I purified water (Milli-Q®, Millipore, Bedford, MA, USA). In some embodiments, the concentration of the cell in the medium may be 9.0×10 ⁸ to 4.0×10 ⁹ cells/mL. This concentration is by reference to the solution in which it is present when introduced into the vessel in which ITP takes place. The concentration of the cells in the medium may be 9.0×10 ⁸ to 4.0×10 ⁹ cells/mL in the TE introduced into the vessel (e.g. the TE in the inlet vial). To further elaborate, the concentration of cells may be determined by reference to its concentration in the electrolyte solution it is combined with for introduction into the vessel in which ITP takes place. This may be the terminating electrolyte (TE) solution, in the case of the cells. The concentration is determined by reference to that solution prior to introduction into the vessel, where it is brought into contact with the other components of the solution to which the voltage is applied in the method of the invention.

In some embodiments, the concentration of the nucleic acid in the medium may be 0.35 to 0.40 mg/uL. This concentration is by reference to the solution in which it is present when introduced into the vessel in which ITP takes place. The concentration of nucleic acid in the medium may be 0.35 to 0.40 mg/uL in the LE composition introduced into the vessel (e.g. the LE in the inlet vial).

The ITP solution may also comprise one or more additives. Suitable additives include a pH modifier, a surfactant, a water-soluble polymer, a further electrolyte and combinations thereof. The additives may be introduced together with any of components (i)-(iv), or with combinations of two or more thereof.

The pH modifier may be selected from an acid, a base or a combination thereof. Suitable acids include mineral acids (such as hydrochloric acid and hydrobromic acid), formic acid, citric acid, phosphoric acid, acetic acid and combinations thereof. Suitable bases include mineral bases (such as sodium hydroxide, potassium hydroxide and ammonium hydroxide), sodium hydrogen carbonate, and combinations thereof. In some embodiments, the pH modifier is a buffer comprising a combination of an acid and a base. Any suitable buffer may be employed, including a phosphate buffer (e.g. phosphate-buffered saline - PBS). Typically, the concentration of pH modifier is from about 0.1 M to about 10M, from about 0.1 M to about 2M or about 1 M.

The surfactant may be selected from any surfactant that does not interfere with the transformation of the cell with the nucleic acid molecule. Suitable surfactants include ionic surfactants (e.g. cationic and anionic surfactants), nonionic surfactants and zwitterionic surfactants. Preferably, the surfactant is a nonionic surfactant. Suitable nonionic sufactants include ethoxylates (e.g. fatty alcohol ethoxylates, alkylphenol ethoxylates, fatty acid ethoxylates, ethoxylated oils and ethoxylated amines and/or fatty acid amides), fatty acid esters of polyhydroxy compounds (e.g. fatty acid esters of glycerol, sorbitol and sucrose), amine oxides, sulphoxides, phosphine oxides and polyethylene glycol (PEG). Typically, the concentration of surfactant is from about 0.1 wt% to about 10wt%, about 0.1 wt% to about 5wt%, or about 0.1 wt% to about 1 wt%.

The water-soluble polymer may be selected from any water-soluble polymer that does not interfere with the transformation of the cell with the nucleic acid molecule. The purpose of the water-soluble polymer is to modify the surface of the capillary in order to suppress the electro-osmotic flow. Only in this way can the cells and nucleic acid molecules move easily to the anode. Suitable water-soluble polymers include polyvinylpyrollidine (PVP), linear polyacrylamide, poly(dimethylacrylamide), polyethyleneoxide, starch (e.g glucomannan) and modified starches (e.g. hydroxyl ethyl cellulose (HEC), hydroxymethyl cellulose (HPC), hydroxypropyl methylcellulose (HPMC) and methylcellulose (MC)). Typically, the

concentration of water-soluble polymer is from about 0.1 wt% to about 10wt%, about 0.1 wt% to about 5wt%, or about 0.1 wt% to about 1 wt%.

The further electrolyte may be any suitable electrolyte other than the LE and TE. Suitable electrolytes include halide salts (e.g. sodium chloride, sodium bromide, potassium chloride, potassium bromide, calcium chloride, calcium bromide, calcium iodide), sodium hydrogen carbonate, and 2-(N-Morpholino)ethanesulfonic acid (MES) and salts thereof. In some embodiments, the further electrolyte is a spacer electrolyte. A spacer electrolyte is an electrolyte comprising an ion that has an ionic mobility between that of the LE and TE. Spacer electrolytes are preferred in embodiments requiring separation of the cell from nucleic acid molecule after transformation. These embodiments will typically be conducted in a vessel comprising a gel that may serve as a sieving matrix to assist in this separation. However, in some embodiments, the solution is free of spacer electrolyte.

Cells

One advantage of the transformation method of the invention is that the cells do not need to be made competent to achieve successful nucleic acid molecule transformation. Any suitable cell may be used, such as a prokaryotic cell or eukaryotic cell. Accordingly, the cells may be non-competent for electroporation and/or chemical transformation. In some embodiments, the cells are competent for EP and/or CT. In other embodiments, the cells were competent for EP and/or CT and have lost their competence to EP and/or CT. In some embodiments, the cells are prokaryotic cells. In one embodiment, the prokaryotic cells are Escherichia coli (E. coli) cells. Any common laboratory strain of E. coli may be suitable. For example, the E. coli cells may be any of the following strains: DH5a, DH10B, DH12S, DHI OBac, PIR1 , PIR2, INVaF, TOP10, OmniMax 2, Mach1 , BL21 , Stbl2, Stbl3, Stbl4, MC1061 , INV1 10, JM109, XL1 Blue, XL10 Gold and combinations thereof.

In some embodiments, the cells are eukaryotic cells. As described in the Examples, the inventors have shown that Jurkat cells can be effectively transformed with nucleic acid using the methods described herein. Jurkat cells are T cells in suspension, and are known to be difficult to transform with nucleic acid using conventional methods. The inventors therefore envisage that the methods described herein may be used to transform any type of eukaryotic cell with nucleic acid. In some embodiments, the eukaryotic cells are eukaryotic cells that can be grown in culture. Examples of eukaryotic cells that can be grown in culture include mammalian cells, yeast cells, insect cells, and plant cells. In one embodiment, the eukaryotic cells are mammalian ceils. Examples of suitable mammalian cells include commonly used cell lines such as Jurkat, HL-60, THP-1 , U937, HeLa, DU145, LNCaP, MCF- 7, H295R, KBM-7, MDA-MB-468, PC3, T-47D, THP-1 , U87, Vero, CHO and MDCK.

The eukaryotic ceils may be cells that are cultured in suspension or as adherent cells. In one embodiment, the eukaryotic cells are cells that are cultured in suspension. Examples of eukaryotic cells that are cultured in suspension include Jurkat, HL-60, THP-1 , U937, and primary lymphocytes. In one embodiment, the eukaryotic cells are cells that are cultured as adherent cells. Examples of adherent eukaryotic cells include HeLa, DU145, LNCaP, MCF- 7, H295R, KBM-7, MDA-MB-468, PC3, T-47D, U87, Vero, CHO, MDCK, and primary fibroblasts.

In some embodiments, the eukaryotic cells are primary cells. Examples of primary cells include lymphocytes, fibroblasts.

The cells are typically dispersed in a cell-carrying dispersion which comprises a carrier and one or more additives. Typically, the carrier will be the same as the medium of the ITP solution. The additives may be selected from an electrolyte, a water-soluble polymer, a surfactant, a dispersing agent and a combination thereof.

The cell-carrying solution may comprise any carrier that is non-toxic to the cells and miscible with the medium of the ITP solution. Typically, the carrier is the same as the medium for the ITP solution. In some embodiments, the carrier is water, preferably purified water. In some embodiments, the carrier comprises the LE and/or the TE.

The dispersing agent may be any agent capable of preventing agglomeration of the cells during the method of the invention. Typically, a strongly polar solvent is used as the dispersing agent, provided that the strongly polar solvent is non-toxic to the cells. In some embodiments, the dispersing agent may be dimethylsulfoxide (DMSO). The dispersing agent may be included in the cell-carrying dispersion in a concentration from 0.001 % v/v to about 10% v/v, about 0.01 % v/v to about 5% v/v, about 0.05%v/v to about 2% v/v or about 0.1 % v/v to about 1 % v/v.

Nucleic acid molecule

The nucleic acid molecule used in the method of the invention may be any nucleic acid molecule suitable for transforming the cell. The nucleic acid molecule may be single- stranded (ss) or double-stranded (ds).

Typically, the nucleic acid molecules are introduced into the vessel in the medium, an amount of cell-dispersing solution, an amount of LE and/or an amount of TE.

It was surprising that the transformation method of the invention provided the increased transformation rate at comparative efficiencies to existing transformation methods for single- stranded nucleic acid molecules (>25 bases) and for double-stranded nucleic acid molecules. In particular, the enhanced transformation rate relative to EP is surprising. The enhanced rate may also enable transformation with nucleic acid molecules not stable under EP or CT methods.

As used herein, the term‘nucleic acid molecule’ refers to DNA and RNA and combinations thereof as well as nucleic acid-like molecules such as those with substituted backbones and/or unnatural nucleotides.

The nucleic acid molecule may comprise a ribonucleic acid (RNA) molecule, a

deoxyribonucleic acid (DNA) molecule or a combination thereof. The nucleic acid molecule may comprise any combination of natural and unnatural nucleotides. Typically, the nucleotides comprise a nucleoside selected from adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U) and combinations thereof. Examples of unnatural nucleotides include pseudouracil, 3-methyluracil, dihydrouracil, 5-alkylcytosines (e.g., 5-methylcytosine), 5-alkyluracils (e.g., 5-ethyluracil), 5-halouracils (5-bromouracil), 6-azapyrimidine, 6-alkylpyrimidines (6-methyluracil), 2-thiouracil, 4-thiouracil, 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5'-carboxymeihylaminomethyl-2-thiouracil, 5- carboxymethylaminomethyluracil, 1 -methyladenine, 1 -methylhypoxanthine, 2,2- dimethylguanine, 3-methylcytosine, 2-methyiadenine, 2-methyiguanine, N6-methyladenine, 7-methylguanine, 5-methoxyaminomethyl-2-thiouracil, 5-methylaminomethyluracil, 5- methylcarbonylmethyluracil, 5-methyloxyuracil, 5-methyl-2-thiouracil, 2-methylthio-N6- isopentenyiadenine, uracil-5-oxyacetic acid, 2-thiocytosine, purine, 2,6-diaminopurine, 2- aminopurine, isoguanine, indole, imidazole, xanthine, etc.

In some embodiment, the nucleic acid molecule comprises a substituted backbone such as

LIMA (locked nucleic acid), ENA (2'-0,4'-C-ethylene-bridged nucleic acids),

peptide nucleic acids (PNAs), or morpholino backboned nucleic acids.

In some embodiments, the nucleic acid molecule will comprise one or more gene(s). In one embodiment, the gene encodes a eukaryotic protein. In one embodiment, the gene encoding a eukaryotic protein comprises introns, exons and regulatory regions, such as a promoter region. In some embodiments, the nucleic acid molecule is DNA. The DNA may single stranded or double stranded. In some embodiments, the DNA is complementary DNA (cDNA). cDNA typically comprises a gene encoding a eukaryotic protein which lacks introns.

In some embodiments, the nucleic acid molecule is RNA. The RNA may be mRNA, dsRNA, tRNA, antisense RNA, CRISPR RNA.

In another embodiment, the nucleic acid may comprise sequences for gene silencing or gene editing. Examples of suitable gene silencing nucleic acids include iRNA, anti-sense DNA and anti-sense RNA. Examples of gene editing sequences include CRISPR/Cas (e.g., CRISPR/Cas9), and TALEN.

The nucleic acid molecule may be linear or cyclic. One example of a cyclic nucleic acid molecule is a plasmid.

When the nucleic acid is a single-stranded nucleic acid molecule, it comprises at least 25 bases. In some embodiments, the single-stranded nucleic acid molecule comprises at least about 30 bases, about 50 bases, about 100 bases, about 150 bases, about 200 bases to about 300 bases. The single stranded nucleic acid molecule may comprise a number of bases between any two of the above numbers, such as from about 25 bases to about 100 bases, about 25 bases to about 200 bases or about 25 bases to about 300 bases.

When the nucleic acid is a double-stranded nucleic acid molecule, it may comprise any number of base pairs (bps). In some embodiments, the double-stranded nucleic acid molecule comprises at least about 5bp, about 10bp, about 15bp, about 20bp, about 25bp, about 30bp, about 50bp, about 100bp, about 150bp, about 300bp, about 500bp, about 600bp, about 700bp, about 800bp, about 900bp, about 1000bp, about 1500bp, about 2000bp, about 2500bp, about 3000bp, about 3500bp, about 4000bp, about 4500bp, about 5000bp, about 5500bp, about 6000bp, about 6500bp, about 7000bp, about 7500bp, about 8000bp, about 8500bp, about 9000bp, about 9500bp, about 10000bp or about 15000bp. Double-stranded nucleic acid molecules may comprise any number of base pairs between those described above, such as from about 5bp to about 15000bp, about 10bp to about 15000bp or about 5000bp to about 15000bp.

Leading electrolyte (LE)

The leading electrolyte comprises an ionic compound and a solvent. The ionic compound comprises an ion having an ionic mobility greater than the nucleic acid molecule and the cell under ITP conditions. This ion is sometimes referred to herein as the“leading coion”. The ionic compound also comprises a counter ion to balance the charge of the leading coion.

To determine suitable leading electrolytes, a simple experiment can be conducted to check the ionic mobility of the ion of the electrolyte ionic compound and to compare this to the ionic mobility of the cells and the nucleic acid molecule under the same conditions.

In general, chloride is an ion with high ionic mobility, and therefore likely to have higher ionic mobility than the nucleic acid molecule and the cell, so chloride and phosphate electrolytes are typically suitable. Examples of suitable ionic compounds for use as the leading electrolyte include tris(hydroxymethyl)aminomethane hydrochloride (Tris HCI), sodium phosphate (Na-Phos), or a combination thereof.

In some embodiments, the leading electrolyte is an ionic compound comprising an ion selected from chloride, phosphate or a combination thereof. In these embodiments, the counter ion may be selected from tris(hydroxymethyl)aminomethane (Tris), sodium (Na ⁺ ) or a combination thereof. The solvent of the leading electrolyte will typically be the same as the medium of the ITP solution. In some embodiments, the solvent is water, preferably purified water.

The leading electrolyte may comprise the ionic compound in a concentration from 1 mM to about 500mM, about 1 mM to about 250mM, about 20mM to about 100mM or about 50mM.

The leading electrolyte may also comprise one or more additives. Suitable additives include a pH modifier, a surfactant, a water-soluble polymer, a further electrolyte and combinations thereof. Any of these additives described herein may be included in the leading electrolyte.

Terminating electrolyte

The terminating electrolyte comprises an ionic compound and a solvent. The ionic compound comprises an ion having an ionic mobility less than the nucleic acid molecule and the cell under ITP conditions. This ion is sometimes referred to herein as the“terminating co- ion”. The ionic compound also comprises a counter ion to balance the charge of the terminating co-ion.

To determine suitable terminating electrolytes, a simple experiment can be conducted to check the ionic mobility of the terminating ion and to compare this to the ionic mobility of the cells and the nucleic acid molecule under the same conditions.

In general, weak acids make suitable terminating electrolytes. An acid with a pKa around 4.5 - 6.8 and preferably around 4.7 - 6.5 may be considered. Examples of suitable ionic compounds include tris(hydroxymethyl)aminomethane 4-(2-hydroxyethyl)-1 - piperazineethanesulfonic acid (Tris-HEPES), tris(hydroxymethyl)aminomethane

cyclohexylaminoethanesulfonic acid (Tris-CHES), sodium 4-(2-hydroxyethyl)-1 - piperazineethanesulfonic acid (Na-HEPES), and combinations of one or more thereof.

In some embodiments, the terminating electrolyte is an ionic compound comprising an ion selected from 4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid (HEPES),

cyclohexylaminoethanesulfonic acid (CHES) or a combination thereof. In these

embodiments, the counter ion may be selected from tris(hydroxymethyl)aminomethane (Tris), sodium (Na ⁺ ) or a combination thereof.

In some embodiments, the counterion of the terminating electrolyte and the leading electrolyte will be the same.

The solvent of the terminating electrolyte will typically be the same as the medium of the ITP solution. In some embodiments, the solvent is water, preferably purified water.

The terminating electrolyte may comprise the ionic compound in a concentration from 1 mM to about 500mM, about 1 mM to about 250mM, about 20mM to about 100mM or about 50mM.

In some embodiments, the concentration of the terminating electrolyte and the leading electrolyte will be about the same.

The terminating electrolyte may also comprise one or more additives. Suitable additives include a pH modifier, a surfactant, a water-soluble polymer, a further electrolyte and combinations thereof. Any of these additives described herein may be included in the terminating electrolyte.

The invention also provides a cell transformed by the method of the invention. The cell may be transformed by any of the methods described herein. In some embodiments, the cell is transformed to express a label, such as a fluorescence emitting molecule. Suitable fluorescence emitting molecules include green-fluorescent protein and fluorescent variants thereof.

The invention provides a kit comprising the components required to carry out the method of the invention.

In one embodiment, the kit comprises in separate parts (a) a leading electrolyte and (b) a terminating electrolyte; and at least one of (c) a nucleic acid molecule and/or (d) a cell.

In another embodiment, the kit comprises in separate parts (a) a leading electrolyte and (b) a terminating electrolyte; and wherein part (a) and/or part (b) further comprises at least one of (i) a nucleic acid molecule and/or (ii) a cell.

The kit may further comprise any additional component used in the transformation method described herein either in a separate part of as part of one of the parts defined for the kit. Examples

The invention will be further described by way of non-limiting example(s). It will be

understood to persons skilled in the art of the invention that many modifications may be made without departing from the spirit and scope of the invention.

Example 1

This example describes the optimization of transforming TOP10 E. coli cells with nucleic acid molecules of various lengths by the transformation method of the present invention, as well as comparison of this method with typical transformation methods.

Experimental

Chemicals. Tris (hydroxymethyl)aminomethane) (Tris), ³ 99.95%, polypyrolidine (PVP),

(MW 1300 kDA), 4-(2-hydroxyethyl)-1 - piperazineethanesulfonic acid (HEPES) 99.5%, dimethyl sulfoxide anhydrous (DMSO) ³ 99.9%, 1 M Tris HCI (pH 8.0), calcium chloride (CaCl ₂ ) anhydrous ³ 97%, glycerol ³ 99.5%, magnesium sulfate (MgSO ₄ ) ³ 99.5%, magnesium chloride (MgSO ₄ ) hexahydrate minimum ³ 99.5% and ampicillin sodium salt were purchased from Sigma Aldrich (St. Louis, USA). Tryptone, yeast extract and phosphate buffered saline (PBS) were purchased from Oxoid (Hampshire, England). Hydrochloric acid (HCI) 37% and D(+)-Glucose anhydrous were purchased from Merck (Darmstadt, Germany). Sodium hydroxide (NaOH) ³ 98% was purchased from Scharlau (Barcelona, Spain), potassium chloride from Chem-Supply (Gillman, Australia), sodium chloride from Univar (Sevenhill, Australia) and agar from Gelita (Beaudesert, Australia). The plasmid pUC18, kanamycin sulfate, SYTO9 Green Fluorescent Nucleic Acid Stain and SYTO59 Red

Fluorescent Nucleic Acid Stain were purchased from Thermo Fisher Scientific - Life

Technologies Australia Pty Ltd. (Scoresby, Australia). The plasmid pSF-OXB20-daGFP was purchased from Sigma-Aldrich (Castle Hill, Australia) and the DNA ladder plasmids (5, 6,8,

10 kb plasmid, ampicilin resistance) were purchased from Gene and Cell Technologies (Vallejo, USA). The Midori Green Advance DNA Stain was purchased from Nippon Genetics EUROPE GmbH (Duren, Germany).

Bacterial strain and preparation for ITP-based transformation. A strain of TOP10 Escherichia coli (Invitrogen) that had lost competency, as judged by the inability of standard protocols to result in transformation, was used. To prepare non-competent E. coli TOP10 cultures, a single colony was inoculated into sterile centrifuge tubes (50 mL) containing 20 mL Luria-Bertani (LB) medium 1 % NaCI medium and grown for 16 hours at 37°C in an orbital incubator at 180 rpm. The cultures were grown to stationary phase with a cell density of 1 -2 x 10 ⁹ cells/mL, 1 mL of cell suspension was harvested by gentle centrifugation for 7 min at 2500 rpm at 4°C (Eppendorf Centrifuge 5417R supplied by Lab Supply Pty Ltd, Sydney, Australia). The supernatant was discarded, and the cell pellet was subjected to washing with 0.5 mL of TE containing 1 % v/v DMSO by gentle vortex for 5 seconds and finally resuspended into 0.5 mL of sterile TE containing 1 % v/v DMSO for ITP-based transformation study.

Capillary electrophoresis. All CE experiments were carried out on a Beckman Coulter P/ACE MDQ Capillary Electrophoresis System equipped with 488-nm laser module and the capillary was maintained at 25°C. An external bench top laser with output at 635 nm (Thorlabs, New Jerseys, USA) was fiber coupled to the second laser port on Beckman Coulter. Experiments were conducted using a bare fused silica capillary (Polymicro Technology, AZ, USA) of 50 mm id with total length of 40 cm (30 cm effective length to detector). For experiments where cells were collected for culture, the laser was turned off.

Electrolytes. For all ITP experiments, the leading electrolyte (LE) used was 50 mM TRIS HCI (pH 8.0) with 0.5% w/v PVP. The terminating electrolyte (TE) was 50 mM TRIS HEPES (pH 7.9). All solutions were prepared using 18.2 MW cm ¹ Type I purified water (Milli-Q®, Millipore, Bedford, MA, USA) filtered through 0.2 mm sterile filters (Milex-GS Syringe Filter, Merck Millipore, Bayswater, Australia).

Capillary conditioning. Prior to use, the new capillaries were pre-conditioned at 20 psi in the following order: 1 M NaOH (30 min), Milli-Q® water (20 min), 1 M HCI (20 min), Milli-Q® water (10 min) followed by 1 % w/v PVP at 45 psi for 45 min. Finally, the capillary was conditioned with LE at - 16 kV for 10 min.

ITP-based transformation. Each analysis began by flushing the capillary with LE solution for 3 min at 20 psi, hydrodynamic injection of DNA (in LE) at 9 psi for 10 seconds (0.1 mg plasmid DNA), hydrodynamic injection of cells (in TE containing 1 % v/v DMSO) at 9 psi for 5 seconds (2-3 × 10 ⁵ cells), and application of -16 kV between the inlet (TE) and outlet (LE) vials.

ITP-based transformation assisted with counter-pressure. Each analysis began by flushing with LE solution for 4 min at 20 psi, hydrodynamic injection of DNA (in LE) at 9 psi for 10 seconds (0.1 mg plasmid DNA), hydrodynamic injection of cells (in TE containing 1 % v/v DMSO) at 9 psi for 5 seconds (2-3 × 10 ⁵ cells), simultaneous application of voltage (-16 kV) and counter-pressure (1 .3 psi) for a specific time, and subsequent removal of the counter pressure. ITP-based transformation success. After each run, the collected bacterial cells in the outlet vials were immediately transferred to ice and 100 mL of S.O.C. medium (2% tryptone, 0.5% yeast extract, 10 mM NaCI, 2.5 mM KCI, 10mM MgCI ₂ , 10 mM MgSO ₄ , 20 mM glucose) was added. The cells were then incubated for 1 hour at 37°C with shake at 200 rpm. The cells from each transformation were spread on pre-warmed selective plates containing 100 mg/mL ampicillin or kanamycin (for pSF-OXB20-daGFP) and incubated O/N at 37°C. The number of transformed cells were determined by plate counting. The transformation efficiency was calculated dividing the number of transformed CFU by the amount of plasmid DNA injected. The transformation rate was calculated based on the percentage of individual cells capable of being transformed.

Preparation of non-competent cells for chemical transformation and electroporation.

Stationary phase cell cultures were washed twice in 0.1 M CaCI ₂ and 15% v/v glycerol for chemical transformation and electroporation, respectively. Cells were concentrated to 1 -2 × 10 ¹⁰ and diluted to 1 -2 × 10 ⁵ for chemical transformation and electroporation.

Plasmid DNA purification and quantification. Plasmid DNA was purified from liquid cultures using UltraClean Mini Plasmid Prep Kit (Mo Bio Laboratories, Inc., Solana Beach, USA) and Pure Yield Plasmid Midiprep System (Promega Corporation, Madison, USA) according to the manufacturers' protocols.

Viability assay. The cell viability was calculated based on the percentage of CFU obtained from the outlet.

Preparation of electro-competent TOP10. To prepare electro-competent TOP10 E. coli, a single colony was inoculated into sterile centrifuge tubes (50 mL) containing 20 mL LB medium 1 % NaCI medium and grown for 16 hours at 37 °C in an orbital incubator at 180 rpm. 0.5 mL of overnight culture was inoculated into a sterile 250 mL conical flask containing 50 mL of pre- warmed LB medium and the culture was grown until OD ₆₀₀ of 0.6. Then, the culture was chilled on ice for 15 minutes and harvested centrifuged at 2500 g at 4°C for 15 minutes (Eppendorf Centrifuge 581 OR supplied by VWR International Pty Ltd, Tingalpa, Australia). The supernatant was then discarded, and the cell pellets were resuspended in the same volume of ice cold sterile water, followed by another wash as previous steps. Finally, the cell pellet was resuspended in 1 mL of sterile 15% v/v cold glycerol and aliquoted into sterile 1 .5 mL micro-centrifuge tubes for electroporation study. Electroporation. To a vial of 100 mL chilled electro-competent cells, 0.1 mg plasmid DNA was added into the cell suspension and mixed gently. The mixture was then transferred using appropriate aseptic techniques to a pre-chilled sterile 0.1 cm electroporation cuvette to avoid the formation of bubbles and electroporated (E. coli Pulser Transformation Apparatus, Bio Rad Laboratories, Pty., Ltd, Gladesville, Australia) at: 1.35kV, 200 W, and capacitor of 25 F, for T 4-5 ms. S.O.C medium was added immediately, subsequently transferred to a sterile 15 mL tube for further incubation at 37°C (200 rpm) for 1 hour to allow cell recovery and expression of the antibiotic resistance genes. Then, 1 to 100% of the cells was plated on pre- warmed antibiotic containing LB agar plate (100 mg/mL ampicillin for pUC18 transformation) and incubated O/N at 37 °C.

Preparation of chemically-competent TOP10. To prepare chemical-competent TOP10 E. coli, a single colony was inoculated into sterile centrifuge tubes (50 mL) containing 20 mL LB medium 1 % NaCI medium and grown for 16 hours at 37 °C in an orbital incubator at 180 rpm. 0.5 mL of overnight culture was inoculated into a sterile 250 mL conical flask containing 50 mL of pre-warmed LB medium and the culture was grown until OD ₆₀₀ of 0.5. Following that, the culture was chilled in ice for 15 minutes and harvested by centrifuged at 2500 g at 4°C for 15 min. The supernatant was discarded, and the cell pellets were resuspended in the same volume of ice cold 0.1 M MgCI ₂ , followed by another centrifugation at the same conditions. The supernatant was discarded, and the cell pellets were resuspended in 25 mL of ice cold 0.1 M CaCI ₂ and left in ice for 1 hour. Another centrifugation was made at the same conditions. Finally, the supernatant was discarded, the cell pellet was resuspended in 1 mL of 0.1 M CaCI ₂ and aliquoted into 100 mL lots in sterile 1 .5 mL micro-centrifuge tubes. All the steps were performed on ice.

Chemical transformation. To a 100 mL chilled chemical-competent cells, 0.1 mg plasmid DNA was added into the cell suspension and mixed gently. The competent cells were kept on ice for 20 minutes, moved to a 42°C thermal bath for 45 seconds, then back to ice incubation for 8 minutes. Then, S.O.C medium was added. The mixture was incubated at 37°C in an orbital incubator at 200 rpm for 1 hour to allow cell recovery and expression of the antibiotic resistance genes. Finally, 1 to 20% of the cells was plated on pre-warmed antibiotic containing LB agar plate (100 pg/mL ampicillin for pUC18 transformation) and incubated O/N at 37°C.

Cells and DNA staining for method validation. Cells were stained by diluting 10 mL TOP10 E. coli cells in a total volume of 100 mL TE containing 1 % v/v DMSO and 1 mM SYTO 9/ SYTO 59. The cell suspension was incubated at room temperature for at least 30 min prior analysis. Plasmid DNA was stained with 1 mL of Midori Green Advance DNA Stain and addition of LE to a total volume of 100 mL.

Toxicity of DMSO on E. coli TOP10 cells. The cells were grown overnight and centrifuged at 2,500 rpm for 10 minutes at room temperature. The supernatant was discarded, and the cell pellet was resuspended in phosphate buffered saline (PBS). The cells were diluted sequentially in PBS to a final concentration of 10 ⁴ CFU/mL. A cell volume of 100 mL was added to tubes containing increasing logarithmic concentrations of DMSO. The mixture was incubated for 60 minutes at 37°C in a shaker at 150 rpm. Subsequently, 100 mL of the mixture was plated in LB plates and incubated overnight at 37°C. The CFU were counted.

Results and discussion

Figure 1 a shows a schematic of the developed ITP method. The leading electrolyte (LE; 0.05 M T ris-HCI) was selected such that the electrolyte anion (chloride) has a higher effective electrophoretic mobility than the terminating electrolyte (TE, 0.05 M Tris-HEPES) anion HEPES. This allows ions and particles with an intermediate effective electrophoretic mobility to concentrate and focus at the moving LE-TE interface (ITP interface). The capillary is initially filled with LE (Figure 1 a.1 ), hydrodynamic injections of plasmid (suspended in LE) and cells (suspended in TE) are made individually in this order, and finally, the inlet vial is switched to TE (figure 1 a.2). When the voltage is applied, an electric field gradient is created at the ITP interface causing plasmids and cells to be concentrated and focused in a small volume at the moving ITP interface (ca 5.9 mm section of the capillary corresponding to ca 1 1 .5 nL) (figure 1 a.3.i). The validation of the ITP method is represented in Figure 1 b where electropherograms show the focusing of plasmid and cells within overlapping narrow peaks at the ITP interface. Plasmid DNA is stained with Midori Green, whereas cells are stained with SYTO59 due to their compatibility with the 488 and 635 LIF channels of the Beckman P/ACE MDQ CE system. The electropherograms of individually injected plasmid and cells at two different concentrations showing their focusing at the same boundary is also represented in Figure 5. Experimental conditions for Figure 5 were as follows: the capillary was flushed with LE for 4 minutes, followed by hydrodynamic injection of the sample suspensions at 9 psi for 15 seconds, switch of the inlet to TE, and separation at -16 kV. The electrophoretic mobilities of plasmid and cells when individually injected were 4.03E-08 m ² V ^-1 s ^-1 (0.55% RSD) and 4.09E-08 m ² V ^-1 s ^-1 (0.20% RSD), respectively. The difference of 0.06 m ² V ^-1 s ^-1 was caused by a slight variance in the electrolyte composition of plasmid and cell suspensions. Within this ITP interface, cells experience an electric field gradient (ca 0.4 kV/cm) that causes cell membrane depolarization creating pores through which the plasmid moves. After migration, the cells are collected from the outlet vial and cultured to assess viability and transformation success through colony screening (figure 1 c).

In initial experiments, 2-3 x 10 ⁴ stationary phase cells and increasing amounts of pUC18 plasmid from 0.0001 to 0.1 mg were used. Stationary phase cells were employed because cells from the exponential growth phase were killed under the ITP conditions. Transformed colonies were observed when 0.1 mg plasmid was used. Then, the amount of plasmid was kept constant and the cell number was increased to 2-3 × 10 ⁵ . This improved transformation efficiency 35-fold (figure 6). To reduce the impact of capillary blockage for higher cell concentrations, 1 % (v/v) dimethylsulfoxide (DMSO) may be added to the cell suspension without significant toxicity (figure 7) and without affecting the cell's effective electrophoretic mobility (figure 8). Cells suspended in 1 % (v/v) DMSO yielded triple the transformation efficiency (figure 6) for high concentrations of cells subjected to the ITP method in a capillary.

To extend the contact time between the cells and plasmid (ca 240 seconds in the previous experiments), a counter-pressure that opposes the voltage-induced ITP movement was applied to hold plasmids and cells within the capillary (figure 1 a.3.ii). When the counter- pressure was removed, the cells migrated toward the outlet. We observed over 4-fold improvements in transformation efficiency and transformation rate at longer counter- pressure times (figure 2). Longer times slightly decreased the cell viability, although it exceeded 60% under all experimental conditions (figure 2). Compared to EP, ITP-based transformation reduces possible harmful effects on the cells including changes in the pH and heterogeneity of the electric field near the electrodes, gas bubbles, and toxic electrode products, and is applicable to non-competent stationary phase cells. This ensures better cell survival and transformation rates.

The developed ITP-based transformation method was compared to conventional EP and CT. For ITP based transformation, cells were prepared as described in methods and denoted as non-competent, whereas electro- and chemically competent cells were prepared for EP and CT transformation, respectively. ITP showed transformation efficiency (figure 3A) ten times better than CT, with no transformed cells observed with EP when using 1 -2 × 10 ⁵ competent cells. The results were even more pronounced when non-competent cells were used, with no transformed cells observed by either CT or EP. When compared to both CT and EP with 1 -2 × 10 ¹⁰ non-competent cells, it was half as efficient as CT and 4 times more efficient than EP, but the ITP used 100,000 times less cells. The ability of the ITP method to transform low numbers of non-competent cells is a great advantage of this method. In terms of transformation rates, our method offers an outstanding performance over CT and EP with improvements of about 3 orders of magnitude (figure 3B). This results from the large excess of DNA molecules over the cell number (ratio of 1 14,974:1 vs. 2:1 in CT and EP) and confining the pUC18 and cells in the narrow ITP band (1 1 .5 nL ).

The effect of plasmid size on transformation success was also investigated. Several plasmids of up to 10 kb were successfully introduced into the E. coli TOP10 cells using the ITP-based transformation with counter-pressure (1 .3 psi) for 21 minutes. The transformation efficiencies were maintained between 8,237 - 1 1 ,677 and the transformation rates were maintained between 0.29 - 0.46 for all plasmids except p10kb. For p10kb the transformation success was 8 times lower than the average (figure 4). The sharp decline of transformation success of E. coli with plasmids above 6 kb is consistent with results from other

transformation techniques, which indicates that it is not a particular limitation of ITP-based transformation but rather a consequence of plasmid dimensions approaching those of the pores.

This Example describes the development of a rapid ITP method for E. coli transformation utilizing a two-electrolyte system, application of constant voltage and low amounts of non- competent cells (10 ⁵ ) and plasmid (0.1 m) at room temperature. This method provided high concentration of extracellular plasmid near the surface of each cell (ratio of 1 14,974:1 vs. 2:1 in CT and EP) improving the transformation rate up to 0.3% for pUC18 (about 1 ,000 higher than CT and EP) with survival rates greater than 60%. A range of plasmids up to 10 kb was successfully introduced into cells.

Example 2

This example describes the optimization of transforming human (eukaryotic) Jurkat T cells with nucleic acid molecules of various lengths by the transformation method of the present invention. Plasmid DNA and Jurkat T cells are sequentially injected into an Rtx™ wax (fused silica) capillary with 60.0 cm total length and 100 mm ID and focused into 18.6 nL by isotachophoresis (ITP) induced by application of high DC voltage (3.0 kV). Through ITP, a large excess of plasmid DNA is brought in contact with the cell surface (1 .48×10 ⁶ plasmid DNA molecules per cell). Jurkat T cells were purposely selected since it transfects poorly under electroporation, offering frequencies which can be insufficient for their applications. Results and discussion

Effect of the voltage in the viability of Jurkat T cells

Jurkat T cells in PBS kept their viability when exposed up to 3.0 kV for 30 seconds and flushed for 2 min at 50 psi through a fused silica capillary of 40 cm length and 50 mm ID (see Figure 9). A voltage of 3.0 kV was settled for the flowing ITP experiments. Optimization of the eletrolytes

For initial experiments, 0.05 M T ris-HCI was selected as leading electrolyte (LE) and 0.05 M Tris-HEPES as leading electrolyte (LE), the established electrolytes for the ITP-based transformation of E. coli TOP10. However, these electrolytes decreased drastically the viability of the Jurkat T cells compromising their focusing by ITP. Therefore, a new buffer composition was formulated containing the dominant ionic species of phosphate buffer saline (PBS), sodium and phosphate, at the same ionic concentration. 0.16 M sodium-phosphate was chosen as LE and 0.16 M sodium-HEPES as TE, such that the anion phosphate has a higher effective electrophoretic mobility than the anion HEPES. This allows the cells with an intermediate effective electrophoretic mobility to concentrate and focus at the moving LE-TE (ITP) interface. The selected LE and TE guaranteed Jurkat T cells viability greater than 80% for more than 10 hours, which was comparable to cells maintained in medium or PBS (see Figure 10). In addition, a concentration of 0.1 % (v/v) DMSO was incorporated in the TE as previously reported for the transformation of E. coli TOP10. This concentration was chosen because it did not affect the viability of Jurkat T cells neither attaches to the capillary wall.

Prediction of the ITP boundary of Jurkat T cells

To predict the ITP interface of the Jurkat T cells (approximately 10 nm diameter) the ITP of fluorescent carboxyl-functionalised microspheres was performed with mean diameter from 0.51 mm and 15.45 mm (see Figure 1 1 ). Under the ITP conditions described in Figure 1 1 , it was inferred that Jurkat T cells could be eluted in less than 35 minutes. Developped ITP method

Late-log phase 7×10 ⁶ -1 ×10 ⁷ cells were place in the inlet vial. To yield a reasonable number of transfectants the experimental conditions described in Figure 1 1 were modified. The length of the capillary was increased from 40 to 60 cm and the inner diameter of the capillary from 50 to 100 mm. Transfected cells were observed when 0.19 mg plasmid was used.

Figure 12 shows the developed ITP method. The capillary is initially filled with LE (see Figure 12 A1 ), hydrodynamic injections of plasmid (suspended in LE) and cells (suspended in TE) are made individually in this order, and finally, the inlet vial is switched to TE (Figure 12 A2). When the voltage is applied, an electric field gradient is created at the ITP interface causing plasmids and Jurkat T cells to be concentrated and focused in a small volume at the moving ITP interface (ca 2.4 mm section of the capillary corresponding to ca 18.6 nL ) (Figure 12 A3). The validation of the ITP method is represented in Figure 12 B where an electropherogram show the focusing of plasmid and cells within the same peak at the ITP interface. Within this ITP interface, cells experience an electric field gradient (ca 0.05 kV/cm) that creates pores in the cell membrane through which the plasmid moves. After migration, the cells are collected from the outlet vial and cultured in 3 ml. non-selective medium. After 16 hours, penicillin- streptomycin is added to prevent bacterial contamination of the Jurkat T cells culture due to their effective combined action against gram-positive and gram-negative bacteria. Protein (GFP) expression is assayed 48 hours after the electroporation procedure by fluorescence microscopy (see Figure 12 C).

Transfection of Jurkat T cells with pCMV6-AC-GFP (plasmid codifying for GFP) via EP and ITP were compared in terms of transfection rate (percentage of cells that were transformed) and transfection efficiency (efficiency of DNA uptake i.e. transfected cells/pg DNA). The ITP- based transfection of Jurkat T cells with the plasmid pCMV6, without expression of GFP, was used as negative control (see Figure 13).

ITP showed transformation efficiency (see Figure 14 A) 396 lower than EP of 3-4 × 10 ⁶ Jurkat T cells, but only twice lower than EP when 1 -2 × 10 ⁴ Jurkat T cells were used. The transfection rate (see Figure 14 B) obtained by ITP transfection was comparable to EP. This can result from a large excess of DNA molecules over the cell number (1 .48×10 ⁶ plasmid DNA molecules per cell) and confining the plasmids and cells in the narrow ITP band (18.61 nL ).

Conclusion

The ITP method of the present invention has been demonstrated for the transfection of human (eukaryotic) Jurkat T cells that only required two electrolyte systems was developed by application of constant voltage (3kV), 1 -2 × 10 ⁴ and 0.19 mg of plasmid DNA. The cell survival under ITP transfection was greater than 8%.