Electropulser: an important tool in Genetic manipulation

By Lam kim Cuong, Ph.D

 

Lời giới thiệu:  Lâm Kim Cương, Ph.D (DKSG68) là giáo sư môn “Molecular Genenitcs and Microbiology” tại Ðại Học Y-Nha khoa New Jersey.  Năm 1987 anh đã sang chế ra máy electroporation đang được nhiều nơi xử dụng để đưa gene mới vào vi trùng và có nhiều công trình khảo cứu về fungus.  Anh cũng là đương kim chủ tịch Cộng Ðồng VN tại NJ.  Ngoài ra còn là một nhạc sĩ có thiên khiếu về sang tác và hoà âm.  Anh đã hợp sức với hai đồng nghiệp Nguyễn Giảng và Lê Văn Thành để sang tác ra bài Dược Khoa Hành Khúc. Trong tiểu sử của anh có nói tới cái máy elctroporation này nên có nhiều thắc mắc.  Do đó chúng tôi đã yêu cầu anh viết một bài để giải thích, bằng anh văn để các đồng nghiệp trẻ có thể theo dỏi dễ dàng.

 

The exploitation of microorganisms for the production of foodstuffs and pharmaceuticals is a common feature of former civilizations. The development of modern industrial microbiology stems from the availability of reliable methods for creating new bacterial strains capable of producing large amount of the desired enzyme (protein) and the growth and maintenance of monocultures in large fermenters. In the pass, strains improvement were done by selection (screening large number of bacteria to find one that can produce a specific compound). It is time consuming and does not necessarily lead to the enzyme we hope to have. The advance in genetics and molecular cloning have make possible to insert a desired foreign gene into bacteria1, mammalian2,3,4 or yeast cells5, 6 and use these new strains as mini factories to generate large quantities of the desired protein. Techniques for isolation and cloning of gene are well documented. However, methods for the introduction of foreign gene into the cell for expression are tricky. A variety of practices has been used to enhance DNA uptake by modification of cell surface (e.g. Calcium Chloride7, Dimethyl sulfoxide8…). The rationales for the effectiveness of these methods remain nebulous. In 1987, I have designed and developed an electrical device (called electropulser or electroporation machine) that uses the high electrical field to introduce DNA into cells (Bacterial, mammalian, yeast cells)9 The electropulsers are now manufactured by different companies. They are frequently used in many laboratories as an efficient tool in genetic manipulation.

 

 

Overview

 

Four different deoxyribonucleotides (Adenine, Cytosine, Guanine and Thymidine) serve as the major components of DNAs. DNA molecules from different cells and viruses vary in the ratio of the four nucleotides monomers, in their nucleotides sequence, and in their molecular weight. Genetic information is stored in DNA, the informational macromolecule of the chromosomes. This information instructs each cell to produce a characteristic set of proteins, in accordance with the central statement of molecular genetics; i.e., genetic information flows in the direction DNA ---> RNA----> Protein

The segment of DNA molecule specifying one complete polypeptide chain (protein) is called a gene

 

Chromosomal DNA

 

Eukaryotic cell (animal, plant, fungi, protozoa, algae) contains a membrane-surrounded nucleus. The genetic material is divided into many chromosomes, each chromosome contains one very large DNA molecule.

 

Prokaryotes (bacteria, blue-green algae, spirochetes, rickettsiae, mycoplasma) have only one chromosome that consists of a single molecule of double helical DNA (containing about 4 million nucleotide pairs), densely coiled to form the nuclear zone.

 

Plasmid DNA.

 

In addition to their single large circular chromosomes, bacterial cells also contain 1 to 20 much smaller circular, double-stranded DNA molecules called Plasmids. They are autonomously replicating. Plasmids carry genes that are not essential for host cell growth, while the chromosome carries all the necessary genes. Plasmids can confer to their host resistance to various antibiotic or synthesis of bactericidal (bacteriocinogens). Because of their small size, these plasmids have been used as vector to introduce foreign genes into bacteria or eukaryotic cells for expression.

 

Chromosomal DNA and plasmid DNA can be easily purified from different cells. Many enzymes (restriction enzyme) can "cut" (digest) DNA at very specific sequences, or “joint" (ligate) two different DNA fragments. These properties are routinely used in genetic manipulation10.

 

Expression of new protein in bacteria:

 

New techniques have vastly expanded the possibilities for creating genetic novelty. Methods for manipulating DNA in vitro have provided the simpler and much broader procedure of molecular recombination in which a segment of DNA from any source can be inserted into a bacterial plasmid. These hybrid plasmids can be multiplied in a bacterial host, expressed and the resulting protein recovered. The extension of cloning to DNA of any source promises to have an extraordinary range of uses, both fundamental and practical. Figure 1a shows a model of expression plasmid (plasmid pKK223-3). The diagram shows many cutting sites (restriction sites) that are recognized by different enzymes, an ampicillin resistance gene that confers resistance to ampicillin once the host bacteria harvest the plasmid. The Promoter region is where RNA polymerase recognizes position to start the synthesis of the inserted gene.

 

For cloning, a foreign gene (e.g. human a-interferon gene) is isolated from the human chromosome DNA by using different restriction enzymes that "cut" (digest) the DNA strand at specific sites then "paste" (ligate) to a plasmid previously digested with similar restriction enzymes. The resulting plasmid will now carry the gene for the new protein and the gene for Ampicillin resistance (fig. 1b)

 

          

 

This newly constructed plasmid has to be introduced into the bacterial cell for expression. This procedure is called transformation. Transformation can occur naturally by mating between cells in contact (conjugation) or by artificial methods (transfection).

 

Classical chemical methods rendering cells susceptible to transformation are often time consuming, tedious and unreliable. A new method using high electrical field pulses or "Electroporation"11, 12 is an excellent solution for introduction of foreign genes into bacteria13, 14, mammalian cells15, 16, plant protoplast17, yeast18,19. Successful transformation will give raise to a strain of bacteria resistant to ampicillin. A single colony is picked and grown in liquid media to a large number of cell (high optical density). The new protein however is not produced until we "turn on" (induce) the gene by adding IPTG (isopropyl thiogalactoside) or by elevating the incubation temperature to 42oC (depending on the type of promoter used, just like we use different type of switch to turn on the motor of a machine). After induction for 1-2 hr, cells are harvested, lyzed (break open) and check for the presence of the protein of interest.

   

Electropration machine (electropulser): Principle and construction

 

The principle of electroporation is based primarily on the action of short electric impulses which above a certain field strength can make biomembranes transiently more permeable yet without permanently damaging the membrane structure20. The electric field pulse is hypothesized to cause disturbance in the phospholipid membrane bilayers leading to pore formation21 (fig. 2). The formation of transmembrane pores causes an increase in membrane permeability, allowing exchange of intracellular and extracellular components.

 

                          

                                          Figure 2

 

A variety of environmental factors such as salt concentration, temperature may also affect DNA transfer. Immediately after the electric field pulse, the transmembrane pores allow an inward transport which is driven by the resultant osmotic pressure leading to cell swelling and, if severe enough, to rupture and lysis.

 

Design of an electropulser:

 

Electric field pulse can be a square wave pulse or capacitor discharge type.

 

A) Square wave pulser:

 

Theoretically a square wave signal can be created by manually turning ON and OFF a constant DC voltage source (Fig. 3a). However for high speed response electronic switches are better. Base on these conditions, a square wave pulser must include a controllable high voltage DC source to provide the high electric field, an electronic timer to set the ON/OFF switch, and an electronic switch capable of reacting to the very fast switching by the timer (Fig. 3b)

 

              

                     Figure 3a                                  Figure 3b

 

Prototype and construction diagram of the LKC square wave pulser are shown in fig. 4 and fig. 5  

 

                                   

    

                   Figure 4                                        Figure 5

 

Due to the complexity of the electronic circuit the construction of this square wave pulser is not recommended for anyone who is not experience in electronic. A simpler version using Capacitor discharge is offered. This machine even very simple in construction has proved to be very efficient in all type of cell transformation.

 

The principle of capacitor discharge type pulser is shown in fig. 6 that includes a power source, a capacitor, a 3 positions ON/OFF/ON switch and a small electroporation chamber to provide high electric field pulse to the cell suspension.

 

Capacitor is a system of conductors that stores energy (like a battery) in the form of electrical field. A capacitor is characterized by its Capacitance C (also called capacity). The potential difference V across a capacitor is always directly proportional to the charge Q on either of it plates: the more the charge, the stronger the electric field between the plated and the greater the potential difference. The ratio between Q and V is therefore a constant for any capacitor and is know as its capacitance (C)

                                        Q (Coulombs)

                            C (farad) =  --------------------

                                        V   (Volts)

 

When a capacitor is connected to a battery (or power source), it becomes "charge" to some value q depending on the resistance R in the circuit. The release of energy stored in a capacitor when a circuit is connected between its terminals is called "Discharge". When a capacitor with an initial charge is discharge through a resistance, its charge decreases with time down to 37% after the time RC where R is the total resistance in the circuit (in Ohms) and C is the capacitance of the capacitor (in Farad).

 

              

                    Figure 6                                         Figure 7

 

The diagram of the CKL Capacitor discharge type pulser (fig. 7) shows capacitors of different C value to provide different time RC for use in different cell type transformation.

 

       

                   Picture 1                           Picture 2                     Picture 4

 

Picture I: the LKC Capacitor Discharge pulser; Picture 2: the chamber holder; Picture 3: Upper, lower plate and the 0.3mm gap isolator of the LKC mini electroporation chamber

 

 

Use of electropulser for bacteria transformation

 

An electrical field of 8000-14000 volt/cm is needed to cause pore formation in bacteria. This high electrical field can only be produced by expensive power supplies that are capable of generating very high voltage to a chamber of 1cm gap. In my electropulse, a small chamber with an electrode gap of only 0.3mm is build. This small gap will help generated a 14,000 V/cm of electric field when only 420 volts is used that is though be large enough for all types of bacterial transformation. Any power supply found in the research laboratory is suitable for its operation. The box holder of the chamber (Picture 2) is made of Plexiglas with cover to prevent any electrical hazard and to provide a safe environment.

 

For bacteria transformation, 10ng to 1mg of the desired plasmid DNA is added to 40 ml of cell suspension. The mixture is then placed in between the sterile aluminum electrode plates. An appropriate electric pulse is applied. Immediately resuspend the pulsed cells in 1ml of regeneration medium and incubate at 37oC for 1 hr. Appropriately dilute cells and plate onto selective agar plates for selection of transformants.

 

The CKL electroporation machine has been successfully used for transformation of bacteria (E. coli ,,aDH5 HB101, LE392, MC1061 and the impossible to transform by chemical method Zymomonas mobilis14), mammalian cells (Human Burkitt lymphoma DAUDI, Human T-cell SUPT-1, human myelogenous leukemia K561, Human peripheral blood HL-60, mouse lymphoid neoplasm P388 D1, mouse lymphocytic leukemia LM1210, and mouse myeloma cells SP2/0 ) and yeast cell ( Saccharomyces cerevisieae) with high efficiency.

 

This machine was donated, as a courtesy gift, to the Microbiology and Biochemistry Department, Cook College, Rutgers University for its routine use in bacteria transformation

 

Conclusion:

 

The success of genetic manipulation largely depends on the ability to re- introduce the newly created gene back into bacteria, yeast or mammalian cells for expression. This can be done by different chemical agents with unreliable effect. The capacitor discharge type electroporation machine described here a simple of low cost and easy to construct yet very effective in all types of cell transformation.

 

 

References:

 

  1. Avery O.T., C.M Macleod, and M. McCarty (1944). Studies on the chemical nature of the substance inducing transformation of Pneumococcal types I. Introduction of transformation by a DNA fraction isolated from Pneumococcus type III. J. Exp. Med. 79:137-158.
  2. Graham, F.L., and A.J. Van der Eb (1973). A new technique for the assay of infectivity of human Adenovirus 5 DNA. Virology. 52:456-467
  3. Szybalska E. H. and W. Szybalski (1962). Genetics of human cell line IV. DNA-mediated heritable transformation of a biochemical trait. Proc. Natl. Acad. Sci. USA 48:2026-2034
  4. Morganelli, C.M. and E.M. Berger (1984). Transient expression of homologous genes in Drosophila cells. Science 224:1004-1006
  5. Beggs, J.D. (1978) Transformation of yeast by replicating hybrid plasmid. Nature 275:104-109
  6. Hinnen, A., Hicks, J.B., abd J.R. Fink (1978). Transformation of yeast. Proc. Natl. Acad. Sci. USA 75:1929-1933.
  7. Mandel, H. and A. Higa (1970). Calcium-dependent bacteriophage DNA infection. J. Mol. Bio;. 53:159-162.
  8. Hanahan, D. (1983). Techniques for transformation of E.coli p.109-135 in: D.M. Glover, DNA cloning: A practical approach. Vol. 1 IRC Press, Washington, D.C.
  9. Lam, C.K. (1990). An inexpensive home made pulser. Transformation of bacterial, mammalian and intact yeast cells. 1st International Conference on Electroporation, Wood Hole, MA. (Abs)
  10. Maniatis, T., E.F. Fritsch, and J. Sambrook (1989). Molecular Cloning A Laboratory Manual. Cold Spring Harbor Laboratories. Cold Spring Harbor, New York.
  11. Neuman, E., K. Rosenheck (1972). Permeability changes induced by electric impulses in vesicular membranes. J. Membrane Biol.. 10:279-290.
  12. Zimmermann, U., J. Schulz, and Pilwat (1973). Transcellular ion flow in Escherichia coli B and electrical sizing of bacteria. Biophys. J. 13:1005-1013.
  13. Zabarovsky, E.R., and Winberg (1990). High efficiency electroporation of ligated DNA into bacteria. Nucleic Acid Res. 18:5912
  14. Cuong K. Lam, Patrick O'Mulan, Douglas E. Eveleigh (1993) Transformation of  Zymomonas mobilis by electroporation. Applied Microbiology and Biotechnology  39:305-308.
  15. Lambert, H., R. Pankov, J Gauthier, R. Hancock (1990). Electroporation-mediated uptake of proteins into mammalian cells. Biochem. Cell Biol. 68:729-734
  16. Raptis, L. and K. L. Firth (1990). Laboratory methods. Electroporation of adherent cells in situ. DNA and Cell Biol. 9:615-621
  17. Jones, H., G. Ooms, and M.G.K. Jones (1989). Transient expression in electroporated Solanum protoplasts. Plant. Mol. Biol. 13:503-511
  18. Hill, D.E. (1989). Integrative transformation of yeast using electroporation. Nucleic Acids Res. 17:8011
  19. Hood, M.T., and C. Stachow (1990). Transformation of Schizosaccharomyces pompe by electroporation. Nucleic Acids Res. 18:688
  20. Benz, R., and U. Zimmermann (1980). Pulse length dependence of electrical breakdown in lipid bilayer membranes. Biochim. Biophys. Acta 597:637-642
  21. Chang, D.C., T.S. Reese (1990). Change in membrane structure induced by electroporation as revealed by rapid freezing electron microscopy. Biophys. J. 58:1-12

 

Câu hỏi anh Lê Văn Nhân:

 

"Tôi nghĩ rằng " square wave pulsor" là dòng điện phát sóng hình vuông ( xem hình vẽ) thay vì hình sin như chúng ta thường thấy. Nếu không đúng, đề nghị anh Cương giải thích thêm"

 

Anh Lâm Kim Cương trả lời:

 

Câu hỏi của anh tuy rất đơn giản nhưng ngầm chứa nhiều ý nghĩa và đã đi đúng vào căn bản của máy electropulser!

Ðúng như anh đã nói, Square wave (pulse) nó thật sự "Square" khác với "Sinewave" có dang Sin (!!!).

Vậy tại sao phải dùng  square pulse mà không dùng Sinewave trong electropulser?

1) Square wave: Cường độ dòng điện sẽ ở vào 1 trong 2 vị trí, hoặc cao nhất (maximum amplitude) hoặc 0. Từ trường (Champ magnetique, electric field) tạo  ra do square wave sẽ đi theo 1 chiều duy nhất làm cho chất DNA  (negatively charged) phải di chuyển với vận tốc rất nhanh. Ảnh hưởng của từ trường này  cũng gây xáo trộn trên màng bao (membrane) của tế bào gây nên những lổ hổng (pores) giúp cho DNA đi xuyên qua được để vào b ên trong tế bào.

2) Sinewave: cường độ dòng điện luôn luôn thay đổi từ 0 đến maximum + rồi trở về 0, rồi lại sang đến maximum -  (I = I max . sinp2FT). Vì từ trường biến đổi mau lẹ qua 2 chiều như vậy nên chất DNA sẽ không di chuyển được, các màng của tế bào cũng không bị xáo trộn. Như vậy kết quả là DNA sẽ không thể xuyên vào tế bào được.