Escherichia Coli Transformation by Heat Shock and Electroporation: A Comparative Study Nikita Shahidadpury Undergraduate Research Thesis University of Florida Department of Chemistry Research Advisor: Dr. Charles R. Martin 7/25/2017 Table of Contents Acknowledgements .......................................................................................................... 3 Abstract ............................................................................................................................ 4 Abbreviations .................................................................................................................... 5 Introduction ....................................................................................................................... 6 Bacterial Transformation ............................................................................................... 6 Chemical Transformation .............................................................................................. 6 Transformation via Electroporation ............................................................................... 7 Low-Voltage Electroporation Device ............................................................................. 8 The Chosen Plasmid Vector: pGFPuv .......................................................................... 8 Methods .......................................................................................................................... 11 Preparation of LB Agar Plates .................................................................................... 11 Preparation of Chemically Competent E. coli ............................................................. 11 Preparation of Electro-competent E. coli .................................................................... 13 Performing Heat Shock ............................................................................................... 14 Performing Plasmid DNA Extraction ........................................................................... 14 Performing Electroporation with a Commercial Electroporator ................................... 15 Performing Low-Voltage Electroporation .................................................................... 16 Results ........................................................................................................................... 19 Controls ...................................................................................................................... 19 Heat Shock Results .................................................................................................... 19 Commercial Electroporator Results ............................................................................ 20 Low-Voltage Electroporation Results .......................................................................... 21 Discussion ...................................................................................................................... 22 Conclusion ...................................................................................................................... 25 References ..................................................................................................................... 26 2 Acknowledgements I would like to express my appreciation to Dr. Charles R. Martin for giving me the great opportunity to participate in research with his group. I would also like to thank Juliette Experton for mentoring me and guiding me through my research project. This has been an invaluable learning experience for me and being a part of Dr. Martin's group has helped me grow as a scientist. I am also thankful to Aaron Wilson for his consistent cooperation and valuable advice. Further, I would like to express my gratitude to Choe Hyunjun and Thinh Nguyen, from Dr. Jon D. Stewart’s laboratory, for aiding with certain procedures in this project and allowing us to use their lab equipment. 3 Abstract Bacterial transformation is a concept that has had a powerful impact on several scientific fields, including medicine, chemistry, and engineering. It involves the incorporation of foreign DNA into bacteria, such as Escherichia coli, in order to express specific proteins or make copies of this DNA. Many methods of transformation exist, and their optimization is key for an efficient transformation. In this thesis, three methods of transformation of E. coli with the green fluorescent protein plasmid vector (pGFPuv) were compared: chemical transformation utilizing a calcium chloride heat shock method, electroporation using a commercial electroporation device, and a low-voltage electroporation device developed by the Dr. Martin group. Our objective was to show that the low-voltage device would result in the greatest yield of transformed bacteria. We successfully transformed E. coli with the chemical and commercial electroporator methods, yielding ampicillin-resistant colonies. The utilization of the low-voltage electroporation device needs further optimization to conclusively state its transformation efficacy. 4 Abbreviations CaCl2 Calcium Chloride DNA Deoxyribonucleic Acid E. coli Escherichia Coli EDTA Ethylenediaminetetraacetic Acid IPTG Isopropyl-1-thio-b-D-galactoside KCl Potassium Chloride KH2PO4 Potassium Dihydrogen Phosphate LB Lysogeny Broth NaCl Sodium Chloride Na2HPO4 Sodium Hydrogen Phosphate NaOH Sodium Hydroxide OD600 Optical Density at 600 nm PBS Phosphate Buffered Saline pGFPuv Plasmid Green Fluorescent Protein SDS Sodium Dodecyl Sulfate SOC Super Optimal Broth with Catabolite Repression UV Ultraviolet 5 Introduction Bacterial Transformation Bacterial transformation is the method by which bacteria uptake foreign DNA and incorporate it into their genome, resulting in genetic variation.1 This can happen naturally in the environment or in laboratory induced conditions.1 Being able to transform the bacterial genome has had a significant impact on many modern technologies, including DNA cloning and genetic engineering.2 Commonly, in molecular biology, Escherichia coli (E. coli) bacteria are used as hosts for expressing or producing DNA.3 The genome for E. coli is well-known and relatively simple, making it the ideal organism to study. Also, E. coli have a fast cultivation rate which allows the experiments to be performed in a reasonable amount of time. Several methods of artificial E. coli transformation exist; the most well-known are chemical transformation via heat shock and electroporation with an electric pulse.2 Both methods will be discussed in this report. Other less commonly known methods include biolistic transformation and sonic transformation.2 Chemical Transformation Chemical treatment of E. coli with calcium chloride, CaCl2, followed by a heat shock (rapid rise in temperature) is the most basic and common method of lab-induced transformation.1 An optimized protocol has been described by Hanahan in 1991 and is currently being followed in biochemistry and molecular biology laboratories.4 It is unclear exactly how the CaCl2 enables the transfer of DNA, but it is thought that the 6 Ca2+ cations help absorb the DNA into the surface of the cell by altering the properties of the components composing the cell membrane.1 The rapid increase in temperature from the heat shock enhances membrane permeability and allows entrance of the DNA into the cytoplasm of the cell. Transformation via Electroporation Electroporation involves the application of a high-voltage pulse in order to increase the permeability of the cell membrane to a foreign substance, such as DNA.5 Electroporation can be a reversible or an irreversible process.5 In reversible electroporation, the pores created in the membrane are temporary, unlike irreversible electroporation which results in permanently open pores and bacterial death. The process of electroporation has been highly developed and is now used today to deliver diverse substances including antibodies, dyes, and drugs.6 Electroporation has also been established as efficient in treating cancer as a means of providing chemotherapy. Furthermore, gene therapy applications, such as vaccination and immunization, have also been developed through the use of electroporation.7 Electroporation is a quick and simple method of bacterial transformation that is accomplished with materials that are commercially obtainable, such as a voltage control device and cuvettes.8 Despite being a much more efficient bacterial transformation method, electroporation has disadvantages that make it less common than heat shock. A very large voltage must be applied to electroporate the cells, which poses a safety concern.9 Also, this very large voltage causes high cell death, mainly due to Joule heating, unfavorable pH, and electric field alterations.7 Additionally, the equipment required for electroporation can be expensive.10 7 Low-Voltage Electroporation Device Prior to my participation in Dr. Martin’s laboratory, the Dr. Martin research group had established a new microscale electroporation device.11 This device utilizes a commercial membrane in which gold microtubes have been deposited. When a voltage is applied to the membrane, a large electric field gradient is created, allowing for E. coli to be electroporated as it flows through the pores of the membrane. Due to the large electric field gradient created in the pores, the voltages utilized are significantly smaller (less than 5 V) than the voltage used in commercial electroporation devices (around 2,000 V). Using fluorescent dyes,