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 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 , such as , 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, the lab was able to demonstrate that 39 ± 2% of

E. coli were successfully electroporated using this device. This value is higher than demonstrated by commercial electroporators (2%). The Dr. Martin group sought to extend the application of this new flow-through electroporation device to the incorporation of a gene in E. coli. I had joined the Martin group to assist with this project.

Our goal was to demonstrate that this new device transforms E. coli with foreign DNA more efficiently than other methods of bacterial transformation. With the help of graduate student, Juliette Experton, and undergraduate, Aaron Wilson, I had spent my first couple months learning how to use this new device as a means of electroporation.

We decided to transform the E. coli via CaCl2 heat shock first to amplify the chosen gene and then extract the plasmid DNA for use in electroporation.

The Chosen Plasmid Vector: pGFPuv

A vector is a DNA molecule that can be used to transfer exogenous genetic material to a host cell or organism.12 It can be a plasmid, i.e. a circular, double stranded

DNA molecule that can be engineered for use in DNA cloning. Plasmid vectors can be

8 inserted into the host bacterial genome and replicated. They are commonly created from recombinant DNA, or combined foreign DNA fragments. This can include an origin of replication, an antibiotic-resistance gene, and an area where the desired exogenous

DNA can be inserted. The origin of replication is a specific sequence of DNA at which plasmid replication commences. Figure 1 displays the method of selecting colonies that have obtained antibiotic resistance.13

Figure 1. The circular plasmid vector contains an antibiotic-resistance gene, insert (desired exogenous

DNA), and origin of replication. Bacteria that are successfully transformed with the plasmid are able to grow on an LB that contains the antibiotic. This figure is from Addgene.13

9 pGFPuv is a variant of pGFP, the green fluorescent protein plasmid vector, which is derived from the jellyfish, Aequorea Victoria.14 Green fluorescent protein (GFP) labeled E. coli fluoresce green, and have therefore been used to track cell populations in aquatic environments.15 pGFP has also been used as a marker to study protein localization and gene expression. The pGFPuv was chosen for its optimal green fluorescence under a UV light (360-400 nm), which is 18 times brighter than pGFP.14 pGFPuv is regulated by the lac promotor which can be induced by isopropyl-1-thio-b-D- galactoside (IPTG). Additionally, this plasmid contains the bla gene which conveys ampicillin resistance. The green fluorescence and the antibiotic resistance were both used as selectable markers to determine if E. coli were successfully transformed. Cells that have not taken up the pGFPuv plasmid will not grow on an agar plate that contains ampicillin. We bought 20 µg of the pGFPuv bacterial expression vector from Clontech to be used for this research project.

10 Methods

Preparation of LB Agar Plates

Culture plates were prepared in 100 mm diameter petri dishes using 35 g/L of LB broth with agar (Lennox) in water. Plates with only LB broth with agar were made to use as control plates to ensure the growth of the bacteria prior to insertion of the plasmid.

Other LB agar plates were prepared containing 100 µg/mL of ampicillin. These plates were utilized to check for the successful incorporation of the pGFPuv vector since the plasmid imparts ampicillin resistance to the E. coli. Additional plates were made containing 100 µg/mL ampicillin and 0.1 mM IPTG. IPTG plates were used to visualize the green fluorescence of the transformed bacteria.

Preparation of Chemically Competent E. coli

The success of transformation is dependent on the proper preparation and storage of the bacteria. A lot of time and care was spent making sure the bacteria were properly prepared for later steps. DH10B E. coli were purchased from ThermoFisher and were prepared for heat shock. 80 mL of liquid LB broth was prepared in a concentration of 20 g/L LB broth (Lennox) in water, and E. coli were inoculated using a plastic inoculation loop. In order to incubate the E. coli at 37 °C, the flask was placed in a water bath with a temperature probe and positioned on top of a Fisher Scientific hot plate set at 37 °C. The entire apparatus was then set on top of a MAXI Rotator movable platform to constantly shake the broth (Figure 2). The E. coli were incubated overnight.

The next day, 1 mL of the previously inoculated broth was added to 160 mL of fresh LB broth. The cells were grown until an optical density at 600 nm, OD600, of about 0.20 was 11 reached, which indicates the beginning of the growth log phase of E. coli. We measured the optical density of the cells using an Agilent 8453 UV-vis spectrophotometer. The flask was placed in ice for 15 min, after which 12 mL of the broth were pipetted into each of six 15 mL centrifuge tubes. The tubes were centrifuged, using an IEC Centra

CL3R from Thermo, at 2 °C, 1100 rpm for 15 min. (All centrifuge steps involved in preparing competent E. coli were performed with these conditions). The supernatant was discarded, and the previous step was repeated to increase the size of the bacterial pellet. 10 mL of ice-cold, 100 mM CaCl2 was added to each tube and the pellet resuspended and placed in ice for 20 min. The tubes were centrifuged, and the supernatant was removed. 5 mL of 100 mM CaCl2 in 10% glycerol was pipetted into each tube, and the bacteria were resuspended. The tubes were aliquoted into 100 µL and placed in 2 mL microcentrifuge tubes. These tubes were stored in the freezer at -

80 °C.

12

Figure 2. In place of a shaking incubator, we assembled this apparatus to culture E. coli in LB broth before preparation into competent cells. It is composed of a Fisher Scientific hot plate set at 37°C with temperature probe on top of a MAXI Rotator.

Preparation of Electro-competent E. coli

W3100 E. coli previously utilized by the lab for electroporation were prepared in deionized water to be transformed by the commercial electroporator.16 The cells were inoculated in LB broth as stated before and cultured overnight. After incubation on ice,

12 mL of the broth were pipetted into each of six 15 mL centrifuge tubes and centrifuged. This step was repeated. The cells were suspended in 5 mL of ice-cold

Ultrapure water obtained from an Aries FilterWorks Gemini filtration system and centrifuged. This step was repeated. The cells were resuspended in 5 mL of ice-cold

10% glycerol in Ultrapure water and centrifuged once more. The cells were again resuspended in 1 mL of 10% glycerol in Ultrapure water and transferred into 2 mL microcentrifuge tubes for storage at -80°C.

13 To prepare cells for our lab’s low-voltage, flow-through electroporation device, we performed the same procedure, however, phosphate buffered saline (PBS) was used in place of the Ultrapure water, and 10% glycerol in PBS was used in place of the 10% glycerol in Ultrapure water. PBS contains 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4 and 1.5 mM KH2PO4 and has a pH of 7.5.

Performing Heat Shock

The heat shock was performed with the help of Dr. Stewart’s lab. 2 µL of GFP plasmid in a concentration of 50 ng/µL and 100 µL of E. coli were combined in a 2 mL microcentrifuge tube.4, 17 The E. coli utilized had been previously prepared in 100 mM

CaCl2 in 10% glycerol. The tube was flicked 4 times to mix the contents. Then, the tube was incubated on ice for 15 min. Following incubation, the bacteria were heat shocked in a water bath set at 42 °C for 45 s. After another 10 min on ice, 50 µL were plated using a sterilized loop on each of two LB plates containing ampicillin, and the plates were incubated overnight (about 14 h).

Performing Plasmid DNA Extraction

We purchased a plasmid purification kit from Qiagen to extract the plasmid obtained after heat shock transformation of E. coli. The procedure was followed as stated by the manufacturer’s directions. A colony that had been transformed by the heat shock was inoculated in 10 mL of LB broth and cultured for 18 h. The next day, the

E. coli were centrifuged, and the pellet was resuspended in a solution containing ethylenediaminetetraacetic acid (EDTA), to weaken the cell membrane.18 A solution containing sodium dodecyl sulfate (SDS) and NaOH was used to solubilize proteins and

14 other unwanted components of the cells, rupture the membrane, and denature the DNA through the breaking of hydrogen bonds. A solution containing potassium acetate was used to re-anneal the strands of the GFP plasmid, leaving the unwanted contents (DNA and proteins) in the precipitate. The tube was centrifuged and the supernatant (with the

GFP plasmid) applied to the elution column. The plasmid was bound to the membrane of the column with a high salt buffer and eluted with a low salt buffer. The plasmid was further precipitated with isopropanol, and the pellet washed with ethanol. 20 µL of Tris buffer was added to final air dried pellet, and the plasmid DNA was stored in the freezer at -20 °C. Only 20 µg of pGFPuv was bought from Clontech, so we performed this procedure a few times to ensure that we would have ample GFP plasmid to use in the electroporation steps.

Performing Electroporation with a Commercial Electroporator

50 µL of electro-competent W3100 E. coli that had been prepared in water was combined with 1 µL of GFP plasmid DNA (extracted from the bacteria transformed by heat shock) and transferred to an electroporation cuvette.16 We electroporated the bacteria using the commercial Bio-Rad E. coli Pulser from Dr. Stewart’s laboratory. The

E. coli were electroporated at 2.5 kV. Immediately after electroporation, the cuvette content was pipetted in 0.5 mL SOC medium and incubated for one hour at 37°C. After incubation, 150 µL of the cells were spread on each of two LB agar plates containing ampicillin. The plates were incubated overnight at 37 °C.

15 Performing Low-Voltage Electroporation

The setup of this device is described in the paper by Experton.11 It involves the use of a 15 mm PermeGear Franz Diffusion Cell. The gold microtube membrane was placed in between a top feed chamber and a bottom receiver chamber, and the apparatus clamped together (Figure 3). A PumpPro pump was utilized to pull the bacterial cells from the feed chamber through the membrane where they are electroporated, and moved into the receiver chamber. Voltage pulses of -4.5 V were applied for 30 ms every 250 ms between the membrane and a platinum wire.

The system used previously in the paper employed fluorescent probes to observe the electroporation.11 Fluorescent probes are relatively inexpensive and easily acquired, therefore a large volume of cells with the fluorescent probe (3 mL) was employed. However, with the GFPuv plasmid, lower volumes are preferred to minimize the amount of plasmid used for each experiment. Consequently, some modifications to the previously described protocol were required (Figure 4). We decided to load 100 µL of electro-competent E. coli and 2 µL of GFPuv plasmid in the feed chamber.

Furthermore, a smaller surface area of the gold microtube membrane was utilized. The membrane (Figure 5) was covered with tape, with a 6.5 mm hole cut to allow the bacteria to flow through at a rate around 1 µL/s. A copper tape was attached to the membrane to secure the clamp connected to the working electrode of the potentiostat.

The cells were flowed through the apparatus for 5 min. While cells were flowing, voltage pulses similar to the ones employed in the previous protocol were applied.11 However, the platinum counter electrode could not be placed in the receiver chamber as previously described because the volume was too low. We decided to place it in the

16 receiver chamber through a sealed opening. We filled the bottom receiver chamber with liquid LB broth. After electroporation, we pipetted 150 µL of the bacteria from the receiver chamber onto an LB agar plate containing ampicillin and incubated overnight at

37 °C. This low-voltage method was performed twice, with a better sealant in the receiver chamber the second time (see Discussion section).

Figure 3. A schematic representation, obtained from the paper by Experton, of the low-voltage electroporation device used and described previously by the Dr. Martin group.11

17

Figure 4. The PermeGear Franz Diffusion Cell utilized for the low-voltage electroporation. The E. coli and plasmid are loaded into the top feed chamber, while the lower receiving chamber is filled with LB broth.

Figure 5. Polycarbonate gold tube membrane which is placed between the feed chamber and receiver chamber of the PermeGear Franz Diffusion Cell.

18 Results

Controls

We used an LB agar plate with no antibiotic as our positive control. The untransformed E. coli strains used in this experiment grew successfully on these plates when incubated at 37 °C, indicating that the strains were fine to use for the project. The agar plates with 100 µg/mL ampicillin served as our negative control. No untransformed bacterial colonies grew on these plates, demonstrating the efficacy of the ampicillin.

Heat Shock Results

The transformation of the DH10B E. coli through heat shock was successful, and colonies were obtained on the LB agar plates with 100 µg/mL ampicillin. 105 counted colonies were obtained on one plate and 239 colonies were obtained on the other plate.

The plates are depicted below in Figure 6.

Figure 6. The LB agar plates containing ampicillin above depict the result of the heat shock transformation.

19 Commercial Electroporator Results

The transformation of the W3100 E. coli using the commercial electroporator had also been a success. 766 colonies had been obtained on one plate and 407 colonies on the other (Figure 7). The number of colonies should ideally be less than 300 in order to obtain a precise count. Since much larger numbers were obtained, we plan on decreasing the volume of bacteria plated. A colony from one of the plates was streaked on another agar plate containing ampicillin and IPTG to induce the fluorescence. When a UV lamp was shined over the plate, the E. coli glowed green, characteristic of effectively obtaining and expressing the GFP gene (Figure 8). This further demonstrated that our transformation had been a success.

Figure 7. The LB agar plates containing ampicillin above depict the result of the transformation utilizing the commercial Bio-Rad E. coli Pulser. A black marker was used to aid in counting the colonies.

20

Figure 8. A colony from the E. coli that had been commercially electroporated was streaked on a plate containing IPTG to induce the green fluorescence. A UV lamp was utilized to visualize the result of the transformation.

Low-Voltage Electroporation Results

The low-voltage electroporation device had not produced any transformed colonies. In the discussion section I have analyzed why this might be the case.

21 Discussion

As we have shown above, we have studied three methods for transforming

E. coli with the pGFPuv plasmid vector. We successfully obtained colonies utilizing the chemical heat shock and commercial electroporation methods. It was expected that these two methods would yield transformed colonies, as both methods are well- established and already optimized for transformation. The results obtained are of only one trial for each method. We did not obtain any transformed colonies using the low- voltage electroporation device. In principle, based on results from this device obtained prior to this experiment, this electroporation method should have resulted in the greatest number of bacteria transformed.11 We utilized a voltage of only -4.5 V with the low- voltage, flow-through device, a dramatically lower value than the 2.5 kV used with the commercial electroporator. The much lower voltage applied to the membrane should decrease cell mortality, leading to an increase in amount transformed.

Further optimization of the low-voltage method is required to successfully transform E. coli. We believe the low-voltage electroporation method was unsuccessful due to the formation of gas bubbles on the gold membrane surface, preventing the bacteria from passing through the tubes. These gas bubbles resulted from the reduction of water at the membrane, which forms hydrogen gas. We applied -4.5 V to the membrane, a more negative value than the standard reduction potential of water (-0.83

V). By utilizing voltage pulses, as opposed to a constant voltage applied to the membrane, we are limiting the formation of these bubbles, but not completely. When performing the previously published procedure,11 we had noticed a few bubbles on the

22 membrane; however, they did not prevent the bacteria from passing through the tubes.

We believe this is because the area of the membrane (circle of diameter 15 mm), and therefore the number of tubes, exposed to the bacteria were much larger. As such, the bubbles were only blocking a fraction of the tubes. In the published protocol, 3 mL of bacteria with fluorescent dye were pumped at a flow rate of 2 μL/s.11 However, when utilizing the pGFPuv plasmid vector, a significantly lower volume was utilized, about

100 μL, and flow rate, 1 μL/s. Therefore, we altered the experimental procedure to expose a smaller area of the membrane (circle of diameter 6.5 mm) to the E. coli. The blockage effect of water reduction was greater; hence the pump was unable to remove the bubbles from inside the tubes. Consequently, the 100 μL of bacteria and 2 μL of pGFPuv were not exposed to the large electric field gradient inside the tubes, and the bacteria were not electroporated.

We performed the low-voltage electroporation method twice with pGFPuv, utilizing the same conditions as stated in the Methods section. After observing the bubbles the first time, we decided to improve the outlet seal of the receiver chamber, from which the receiver solution is pumped to the outside. With this modification, we hoped that the pump would remove the bubbles from the membrane. Unfortunately, this was not effective. To prevent these bubbles, we would like to try to use shorter voltage pulses (from 30 ms to 10 ms) and a larger membrane area exposed to the cells in the future.

The utilization of heat shock and commercial electroporation methods both resulted in ampicillin-resistant colonies and, therefore, I am able to compare them. The heat shock was a safer method compared to electroporation, which makes it a method

23 of choice for teaching laboratories. The high voltage used for electroporation, 2.5 kV, can be very dangerous, and therefore the instrument needs to be handled with extreme care. Also, performing the heat shock did not require any specific instrumentation, unlike electroporation. However, the preparation of competent bacteria for heat shock was more complex compared to the preparation of cells for electroporation, as it required an additional chemical, CaCl2. The process of chemical transformation also took longer, around 30 min, due to the periods of incubation in hot water bath and on ice. Performing a commercial electroporation transformation only took a few seconds and had a simpler procedure when compared to chemical transformation. It is easy to understand why this method has become a valuable tool to the scientific community and medical field.

There is much work that needs to be done in the future to conclusively state the overall result of this project. The low-voltage method needs to be re-evaluated and tested to see as to why no colonies were produced. The optimization of this method is of great significance, as it utilizes a significantly lower voltage than commercial electroporation, rendering it a safer process.

A gel electrophoresis of the extracted GFP plasmid could be performed in the future to further substantiate the effective transformation of the E. coli. Also, each procedure should be repeated so that the data can be quantified by calculating the transformation efficiency of each method.

24 Conclusion

When I had first joined the Dr. Martin group, I was intrigued by the low-voltage electroporation device that had been developed and its potential application in DNA cloning. Through this project, I learned how to utilize this device and explore the more common chemical heat shock and commercial electroporation methods of transformation as well. Bacterial transformation is a significant area of breakthrough scientific research with numerous applications. I am very grateful for having been awarded the opportunity to study this unique phenomenon, and participate in an exceptionally rewarding educational experience.

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