The Chemical Engineering Journal, 56 (1994) B75-B77 B75

Construction of a low cost and simple electroporator for high transformation efficiencies in E. coLi strains

Lakshmi Prasanna”, K. Venkatesh Gopal”, E. Sivamani”, S. Renganarayananb, K. Udhayakumarc, V. Mohanb** and Kunthala Jayamman” “Centre fm Biotechnology, Anna University, Madras 600 025 (India) bDepartment of Chemical Engineering, Alagappa College of Technology, Madras 600 025 (India) cDepartment of Electrical Engineering, College of Engineering, G%&d~, Madras 600 025 (India)

Abstract

The design and construction of an inexpensive exponential decay pulse generator is described. A new cuvette and electrode design with an interelectrode gap of 1 nun allows sample volumes as low as 25-50 fi to be electroporated. The reduction in the inter electrode distance enhanced the electrical field strengths with low voltage power supplies. The combination of the cuvette and electrode improved the transformation efficiency of E. coli to 10s-lO’o transformants per microgram of DNA with 80% of the surviving population recovered as transformants.

1. Introduction

Electroporation is an increasingly popular and advantageous method for introducing DNA into a wide range of systems where conventional trans- formation techniques are not successfully applied, allowing high transformation efficiencies [ 1,2 1. We have designed a simple, low cost electroporator capable of delivering high voltage in the form of a spike pulse which decays exponentially with time. The device was constructed and tested to transform certain well-established systems such as E. coli. The actual design allows four different pulses of varying time constants. The electrical parameters that give efficient transformation were standardized. The device provides conditions which allow high efficiencies of stable transformation with E. co& similar to that of any other complex commercial electroporation units. The significant improvement of the custom built electroporator is the construction of a specially designed concentric electrode with an interelectrode gap of 1 mm and a sample cuvette that allows sample volumes as low as 25-50 ~1 to be electroporated, which effectively doubles the effective field strength with low voltage power sup- plies (500 V DC). The cost of the custom built electroporator is only Rs. 2000 (approximately US$

*Author to whom correspondence should be addressed.

loo), considerably less than the expensive com- mercial units.

2. Materials and methods

2.1. Construction The electronic components were obtained from

Texonic Instruments, Madras, India:

Simple printed circuit board 1 1 k/l W resistors 2 Electrolytic grade 2 pF, 450 V DC capacitors 12 Toggle switch 1 500 mA ammeter 1 Perspex sheet for cover 1 5ft red 4000 V test lead wires 2 Sft black 4000 V test lead wires 2 B-way rotary switch 1

The circuit is point to point wired according to the diagram of a simple printed circuit board in Fig. 1. The 2 PF capacitors were connected in parallel so that four different capacitances, 2, 4, 6 and 12 pF, corresponding to four different time constants respectively, can be chosen for the ap- plication of a field pulse.

The input leads are connected from a standard power supply (Hoeffer 500 V DC) to the electro- porator. Charging can occur only when the toggle switch is in the open position and the chosen capacitor can be safely and quickly discharged at

0923-0467/94/$07.00 0 1994 Elsevier Science S.A. All rights reserved SSDI 0923-0467(94)06074-6

B76 L. Prasanna et al. / Con~tm&icm of a low cost and simple electroporator

Fig. 1. Circuit diagram of the pulse generator: Rc, charging resistor; Rd, discharging resistor; SW1 , two-way toggle switch; SW2, four-way rotary switch; A, ammeter; EC, electroporation cuvette; Cl-4, electrolytic capacitors in the range 2,4,6 and 12

WE.

I

0

T

EC

-6mm __CI Fig. 2. Cut view of the concentric ring chromium plated brass electrode: I, inner electrode; 0, outer electrode; T, teflon in- sulation; EC, glass electroporation cuvette.

any time through the 1 K resistor by closing the toggle switch. Because the current is instantaneous the resistance of the 500 mA ammeter is charged so that it acts as a voltage meter displaying the voltage across the capacitor. Closure of the toggle switch discharges the 12 PF capacitor and the contacts of the capacitor are then conducted through the output leader to the electrodes of the electro- poration cuvette containing the mixture of cells and DNA to be electroporated. After the construction the circuit was tested with a storage oscilloscope for measuring the exact output current at various time constants.

A volume of 25-50 ~1 of a mixture of cell sus- pension and 100 ng ml- ’ plasmid was electroporated in a chilled electroporation cuvette with a single pulse by charging the 12 PF capacitor at 4500 V cm-’ and discharging it across the electroporation cuvette. The cells were transferred by micropipette to 1 ml SOC medium [4] and allowed to recover for 60 min at 37 “C. After 60 min appropriate dilutions of the electroporated sample were plated on LB agar with ampicillin. Suitable controls were maintained separately.

3. Results and discussion

The electroporation chamber is a 6 X 6 mm2 glass The possible usefulness of electroporation for cuvette with special radius ring (concentric) chro- transformation of Gram negative bacteria using mium plated brass electrode which fits perfectly HBlOl and DH5cu strains ofE. coli was investigated. into the cuvette (Fig. 2). The electrodes are separated The transformation efficiencies by the classical CaCl, by a spacing of 1 mm. The sample cuvette and the method were of the order of 105-lo6 (Table 1). An electrode are thoroughly sterilized by rinsing with array of 2 PF capacitors with 1000 Q resistance 70% ethanol followed by brief exposure to a flame connected in parallel in the order 1,2,3,6 resulted and then cooled by dipping into sterile ice cold in a discharge of 2,4,6 and 12 PF respect.ively with buffer. a maximum field intensity of 4.5 kV cm-’ in a

2.2. Bacterial and plosmid strains The strains of E. coli (K 12) HBlOl and DH5a

were obtained from laboratory stock. The plasmid DNA used in this study was pUC19, obtained from New England Biolabs., Beverly, MA.

2.3. Chemical transformation Chemical transformation of the E. coli strains

with the plasmid DNA was performed according to the method proposed by Mandel and Higa [3].

2.4. Electroporation of intact bacterial cells (E. coli DH5a and HBlOl) and stabLe esgression of the ampicillin resistance marker in transformants

Introduction of the plasmid DNA into intact bac- terial cells using an electroporator of the design described above resulted in stable expression of an antibiotic resistant marker in transformants. To test the system, pUCl9 plasmid, which carries an am- picillin resistance marker, and the DH5a strain of E. coLi were used. The cells were grown in 11 LB [4] to an OD 600 of 0.5-0.8 and harvested by centrifugation. The pelleted cells were washed twice with 1 1 of 1 mM Hepes (pH 7.0) followed by another wash with 1 1 of 20% glycerol. The cells were resuspended in 20% glycerol to a cell concentration of 3 X 10” viable cells per mililitre. A 100 mg ml-’ pUC19 solution was used, obtained by diluting a 1 mg ml-’ stock pUCl9 solution in TE buffer (10 mM Tris pH 8.0; 1 mM EDTA).

L. Prosannu et al. / Construction of a low cost and simple electroporator

TABLE 1. Transformation efficiency of E. coli strains by CaCl, method

I377

DNA (pUC19) concentration (fig)

0.5 1.0 2.0

HBlOl

Frequency

6.2 x lo6 5.1 x 105 4.8x lo5

Efficiency

5.8 x 106 4.7 x lo5 2.7x lo5

1)H5c~

Frequency

7.5 x 106 2.4x lo5 1.8X lo5

Efficiency

3.7x loa 2.1x105 1.5x lo5

TABLE 2. Efficiency of transformation in E. coli stra.ins by electroporation using the newly constructed pulse generator

Capacitance (mA)

Time constant (ms)

DNA (pUCl9) concentration (pg)

Transformation efficiency

HBlOl DH5a

2 2 1 4 4 1 1.3x lo9 1.9x 109 6 6 1 1.8X 109 2.6x lo9

12 12 1 2.4 x lo9 4.1 x 109

normal 500 V DC electrophoretic power pack. Pulse duration was varied by selecting three different capacitors. Use of 2,4,6 and 12 PF capacitors resulted in a time constant of 2,4,6 and 12 ms respectively and the transformation rates under these conditions are represented in Table 2.

No transformants were obtained by discharging 2 PF capacitors, whereas charging 12 PF capacitors to a set voltage of 4.5 kV cm- ’ and discharging through the sample cuvettes gave the best trans- formation rates.

The new cuvette and electrode design with an interelectrode gap of 1 mm allowed sample volumes as low as 25-50 $1; this thin layer electroporation with low resistance causes a pulse duration too brief for electroporation to occur. To solve this problem some modifications were introduced in the cell preparation for the electrotransformation pro- tocol. 20% glycerol was used as electroporation medium to increase the resistance of the sample after washing the cells three tunes with glycerol. The construction of this cuvette and electrode im- proved the transformation efficiency of E. coli to 108-10’ transformants per microgram of DNA.

The design of sample cuvette (6X6 mm’) and the dimensions of the concentric ring chromium plated brass electrodes permits economical use of cells and DNA. The concentric electrode offers more uniform electric fields compared to parallel plate electrodes such that all the cells are subjected to the effect of the field pulse. The coaxial configuration of this electrode with high divergence gives a well- defined field configuration. Typically for E. co&

with plasmid DNA, a 4.5 kV cm-’ gradient is used. For safety reasons kilovolt power supplies were avoided and the high voltage pulse transfection was carried out with lower voltages and smaller gaps. The voltage gradient has to be sufficient to polarize the cell membranes for electroporation to occur. The voltage gradient is a function of the voltage used to charge the capacitor and the electrode separation which is commonly 2-4 mm [ 51. Hence a power supply of a few hundred volts can be used if the electrode separation is small (of the order of 200-500 pm). The concentric electrode was designed in such a way that the inter electrode gap was minimum (1 mm) so that the field strength increases for a given power supply.

Acknowledgement

The authors thank Mr. Balakrishnan of SIBA Elec- tricals, Madras, India for fabricating the electrode used in this study.

References

E. Neumann, M. Schaefer-Rider, Y. Wang and P.H. Hof- Schneider, EMBO J., I (1982) 84 1. U. Ziiermann, Biochem. Biophys. Acta., 694 (1982) 227. M. Mandel and A. Higa, J. MoZecuZur Biol., 53 (1970) 159. T. Maniatis, E.F. Fritsch and J. Sambrook (eds.), Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Lab- oratory, New York, 1982. GA. Hoffiann and GA. Evans, TEEE. Eng. Med. Biol., 5

(1986) 6.