Multiple scientific disciplines are still trying to determine how life began. Although
competing theories on the origins of life on Earth differ in many aspects, they all agree that
the genetic makeup of organisms is adapted to the environment in which they live by the
forces of natural selection; this process is known as evolution. We know that single‐cell
organisms existed before multi‐cell organisms and that cells without a nucleus (prokaryotes)
existed before cells with a nucleus (eukaryotes).
Up until the 1990s, it was widely assumed that the prevailing source of innovations in
evolution are mutations occurring during cell division and thus transferred to daughter cells
(vertical gene transfer). This theory collapsed when scientists began to analyze the
relatedness of organisms by looking at the similarities of their genomes (a process called
phylogenetic analysis). They discovered that tracking the similarities of different genes can
lead to different branching diagrams of relatedness (phylogenetic trees). Genome studies
have also shown that some organisms contain a gene that is absent in their close relatives,
but present in identical or only slightly altered form in some evolutionarily very distant
organisms. These findings implied that the genetic material is not only inherited from the
parent cells, but can also originate from the surroundings and from other organisms. This
process is known as horizontal gene transfer (HGT). The results of phylogenetic studies show
that HGT has been an important source of innovation for evolution that enabled a faster and
more diverse development of early life.
The scientific literature recognizes three mechanisms of HGT: natural competence,
conjugation and transduction. All of the stated mechanisms are biological and are based on
proteins, each with a highly specific function, which implies that these mechanisms are
themselves products of evolution and had thus only occurred during a certain stage of the
evolutionary history. Consequently, we are left with the question whether there exists a
mechanism, perhaps based on simpler physical principles, that could have acted ever since
the dawn of life. One of the most promising such mechanisms is electroporation.
Electroporation is a phenomenon that enables the entry of exogenous matter into
prokaryotic as well as eukaryotic cells. As a laboratory method it was developed four
decades ago and is based on short‐term exposure of cells to a sufficiently strong electric
field. The field is usually created by delivering voltage pulses to a pair of electrodes between
which the cells are positioned. The result of exposure to such pulses is increased
permeability of the cell plasma membrane, which enables the entry of a wide range of
molecules, including DNA, from the environment to the cell, as well as release of such
molecules from the cell into the environment. If the outflow from the cell is not too strong
and the cell survives the exposure to the pulses, this phenomenon is termed reversible
electroporation, otherwise it is known as irreversible electroporation.
In natural habitats hit by a lightning stroke, the electric field in the ground near the
lightning’s point of entry is sufficient for electroporation; very close to that point the
conditions are those for irreversible electroporation and hence release of DNA, while in the
adjacent region in the downward and outward direction the conditions for reversible
electroporation are met, and hence for uptake of DNA.
To assess electroporation as a natural mechanism of HGT, it is necessary to conduct
biological experiments, where in controlled laboratory conditions we strive to come as close
as possible to emulating natural conditions of lightning striking the ground. For this purpose,
we needed to develop a setup allowing such experiments.
The analysis of the abovementioned findings and motivations for this dissertation are
followed by the description of design, construction and testing of a modular system for
lightning exposures (Scientific Emulator of Evolutionary Lightning, with the acronym ZEVS in
Slovene) and the corresponding high‐voltage generator. The ZEVS system allows to expose
biological samples (cells or tissues) in a controlled environment (precisely determined length
of the discharge arc, monitoring the time course and amplitude of the electric current
flowing through the sample, filming the experiments with a high‐speed camera) to
electrostatic discharges with adjustable amplitude of electric current (up to several hundred
amperes). This provides a reproducible emulation of electrostatic discharges that occur in
natural lightning strokes. The system allows the researchers to use an arbitrary generator of
electrostatic discharges with an adequate receiving (ground) electrode and an adjustable arc
length. The modular design of the system enables quick assembly and disassembly, as well
as simple and thorough cleaning.
For the development of the system, we used computer modeling, where we designed and
analyzed the entire system virtually before buildng the first actual prototype. The
dimensions of the system were determined iteratively using numerical calculations of the
distribution of electric current and field based on the finite elements method. The system
was designed such that it was easy to assemble and disassemble, facilitating transport and
thus allowing to conduct experiments in different laboratories. We also paid attention to
allow for the system to be cleaned simply and thoroughly, which substantially decreases the
risk of contamination, while allowing for the reproducibility of experiments. As a material
for components that are required to be nonconductive, we chose polyethylene. For
components where non‐conductivity as well as transparency was required, we used
Plexiglas. Electrodes were initially made of copper, but we discovered that the electric
discharges caused substantial corrosion of such electrodes, so we later replaced copper with
stainless steel, which turned out to be sufficiently resistant to corrosion caused by electric
discharges. For the ground electrode, which is in direct contact with the biological sample,
the choice of stainless steel proved additionally advantageous as it is less susceptible to
electrolytic dissolution and thus results in a much weaker contamination of the biological
sample by the metal ions.
In the first experimental trials of the ZEVS system, we modified a commercial electric Taser
and used it as the electric discharge generator, yielding a discharge current that lasted
several hundred nanoseconds. Later, we designed and constructed a high‐voltage electric
generator that delivers arcs by a controlled 5 kV discharge of a 1 μF capacitor (the ZEVS
generator). Compared to the Taser, the ZEVS generator discharge current was much closer
in its time course to an actual lightning stroke (zero‐to‐peak time of ~5 μs followed by
exponential decay from the peak with a time constant of ~75 μs, corresponding to a peakto‐
half time of ~100 μs). The first biological experiments were conducted on Escherichia coli bacteria planted on agar in petri dishes having inner diameter of 86 mm, with discharges generated by the Taser. Petri dishes with agar and the plated bacteria were inserted into the ZEVS system, the discharge was delivered from the conical electrode, entering vertically downwards into the center of the petri dish, and we supplied 10 consecutive such discharges. The current of each discharge had the peak value of ~100A, zero‐to‐peak time of ~0.1 μs, and peak‐to‐half time of ~0.3 μs. The length of the arc of each discharge was ~15 mm. The experiments produced a circular region of radius of 4 mm from the center of the petri dish in which there were almost no detectable colonies E. coli. The calculated electric field strength at that radial distance was ~8 kV/cm. The acquired results together with these calculations imply that the region devoid of viable bacteria was due to their irreversible electroporation.
The second set of biological expeiments was conducted on Chinese Hamster Ovary (CHO)
cells, which are eukaryotic, again using the Taser to generate the discharges. CHO cells were
plated in petri dishes having inner diameter of 52 mm. Before exposing the petri dishes to
the discharges, we removed the original culture medium and then added 1.5 ml of a fresh
culture medium containing 4 μg/ml plasmid DNA pEGFP‐N1 that contains a gene encoding
the green fluorescent protein (GFP). We then placed the petri dishes into the ZEVS system
and exposed each dish to 10 electrostatic discharges. The electric current of each discharge
had a peak value of ~14 A, zero‐to‐peak time of ~0.5 μs and peak‐to‐half time of ~1.5 μs.
The length of the electric arc in each discharge was ~7mm. On the area spanning radially
from 3 to 15 mm from the center of the petri dish, we detected GFP fluorescence, reflecting
uptake of pEGFP‐N1 and its expression, and thus corresponding to the area of reversible
electroporation. By calculattion, we estimated the electric field strength at 15 mm from the
center of the petri dish as 1.11 kV/cm, and at 3 mm as 5.54 kV/cm. This suggests that the
central region with no gene expression was subject to irreversible electroporation and thus
cell death, while in the outer region in which there was also no detectable expression the
cells were not electroporated, and thus there was no DNA uptake.
The third set of experiments was irreversible electroporation on bacterial spores of Bacillus
pumilus planted on agar in petri dishes. For these experiments, we used the Taser
generator, as well as the ZEVS generator that we had already developed at that stage. With
both discharge generators we achieved reproducible inactivation of the spores. With
experiments utilizing the Taser, we achieved inactivation in 0.65% of the entire petri dish
after delivering 20 electric discharges. Using the ZEVS generator, the area of inactivation
was 7% using one discharge, 27% after 10 discharges, and 55% after 50 discharges.
The conducted experiments have shown that the ZEVS system is suitable for studying the
effects of discharges on both prokaryotic and eukaryotic cells, and that with it we can
achieve irreversible electroporation that causes leakage of DNA, as well as reversible
electroporation that results in uptake and expression of DNA. Experiments of irreversible electroporation in E. coli and of gene uptake in CHO cells suggest that electroporation could act as the fourth natural mechanism of HGT. To arrive at a reliable and quantitatively relevant answer, however, it is necessary to conduct further experiments on organisms whose natural environment is accessible to lightning strokes (e.g. bacteria populating the top layers of seawater and freshwater habitats). Furthermore, for reliable conclusions it is important to use natural DNA, devoid of artificial modifications often present in commercially available DNA with the aim to increase its stability and/or the efficiency of uptake and expression.
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