Volume 3
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Number 2
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June 2010
10.1093/biohorizons/hzq018
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Research article
The effect of ultrasound exposure on the
transformation efficiency of Escherichia coli HB101
Kimran Hayer*
University of Derby, Derby, UK.
* Corresponding author: School of Biology, University of Nottingham, University Park, Nottingham NG7 2RD, UK. Tel: þ44 115 95 18479. Email: [email protected]
Supervisor: Dr Ian Turner, University of Derby, Kedleston Road, Derby, DE22 1GB, UK.
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Transformation is an important tool in modern genetic engineering and artificial methods exist to induce transformation in bacteria.
Ultrasound offers the potential advantage of being versatile and less dependent on cell types than traditional methods like electroporation. This study investigated the effect of low-frequency ultrasound exposure on the ability of Escherichia coli (E. coli) to undergo
transformation. E. coli HB101 in the presence of pBR322 plasmid was exposed to ultrasound frequencies of 48 kHz for 10 – 1200 s
and monitored over a 24 and 48 h period. The most effective transformation efficiency (148.72 transformants mg21 of DNA) was
observed at 10 s exposure to ultrasound and after 24 h incubation. The ultrasound method was compared with the calcium chloride
(CaCl2) method of inducing artificial competence. There was a significant difference between 0.05 mM CaCl2 induced transformation
(4.70 transformants mg21 of DNA) and 10 s exposure to ultrasound transformation (148.72 transformants mg21 of DNA) after 24 h incubation. This study highlights the potential of ultrasound as a realistic alternative to induce competence for the genetic manipulation of
bacteria.
Key words: Escherichia coli HB101, transformation, ultrasound, calcium chloride, transformation efficiency.
Submitted on 15 September 2009; accepted on 7 April 2010
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Introduction
Transformation is one of the key mechanisms in which bacteria are capable of DNA transfer.1 It refers to the mechanism by which bacteria uptake naked DNA from the
environment across their cell membranes and incorporate it
into their genomes.2 – 5 This mechanism of DNA acquisition
often has significant implications, whereby bacteria are able
to pick up and acquire advantageous traits like antibiotic
resistance.6, 7 Some bacteria like Bacillus subtilis are naturally capable of transformation and are regarded as ‘genetically competent’.8 Escherichia coli, however, is a bacterial
species that is not capable of undergoing natural transformation and so requires some sort of artificial intervention to
perform this process. The treatment of non-competent cultures like E. coli with chemical or physical agents can
permit the uptake of DNA, via the induction of artificial
‘competence’ under laboratory conditions.1, 9
Ultrasound has been identified as a potential novel technique for affecting transformation because it is recognized
as a method responsible for causing biological effects.
Ultrasound refers to waves which can transport mechanical
energy through local vibration of particles at frequencies of
20 kHz or more.10 Ultrasound is classified into: power
ultrasound (20–100 kHz) used for sonochemistry and highfrequency ultrasound (1 –10 MHz) used for diagnostic purposes.11 Power ultrasound may disrupt the lipid membrane
to have an impact on how bacteria can grow and be transformed—either by facilitating a higher degree of transformation across the bacterial cell population, or hindering the
process altogether. Progress in the understanding of bacterial
genetics is dependent on the availability and development of
transformation methods12 thus ultrasound may provide a
means of inducing artificial competence.
This novel research parallels a study carried out by Song
et al.13 who investigated ultrasound-mediated DNA transfer
using Pseudomonas putida UWC1, E. coli DH5a and
Pseudomonas fluorescens SBW25 as recipients for the
plasmid pBBR1MCS2. To investigate the efficiency of
ultrasound-mediated DNA delivery, comparisons were made
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#
The Author 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons
Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use,
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with other transformation mechanisms—namely electroporation and conjugation. A key finding from this study was that
using an ultrasound duration of 10 s, 50 mM CaCl2, at a temperature of 228C and a plasmid concentration of 0.8 ng/ml, an
efficiency of (9.8 + 2.3)*1026 transformants per cell was
achieved in P. putida UWC1.13 This was found to be nine
times greater than conjugation and four times more efficient
than electroporation.13
Transformation as a method of bacterial gene transfer
Transformation is a process that is seen widely across the
bacterial
kingdom5
both
in
Gram-positive and
Gram-negative bacteria e.g. B. subtilis and Neisseria gonorrhoeae, respectively.6, 14, 15
A large number of bacterial species are either not amenable to being cultured or are not suitable for traditional
culture-dependent DNA delivery methods.13 With bacteria
that can be cultured in vitro, there is a lack of efficient
methods for genetic manipulation,13 so the application of
ultrasound is potentially useful. Laboratory methods of inducing transformation are described below.
Electroporation
Electroporation refers to the transfer of DNA through membrane pores formed via high-voltage electric fields.9, 16 – 20
This is a rapid and simple procedure, where essentially a
high-voltage is applied to a suspension of cells with DNA
placed between electrodes in a cuvet.12, 21 Calvin and
Hanawalt22 used electroporation to generate pores in the
outer membranes of E. coli K12 derivatives, which were
large enough and persisted long enough to enable gene
flow. This led to transformation of bacterial cells with an
efficiency of 109 transformants mg21 of plasmid.22
Song et al.13 used the electroporation technique and compared it to ultrasound treatment.
Calcium chloride
This is a long used transformation method 9, 18 due to the
observation made in the 1970s when it was found that
E. coli cells soaked in ice-cold salt solution were more efficient at DNA uptake than the untreated cells.23 Thus, a
key breakthrough was made from the initial idea that such
species was refractory to transformation24 when Cohen
et al.25 found that CaCl2 treated cells made more effective
recipients for plasmid DNA. In almost all Gram-negative
bacteria which have been transformed, the use of CaCl2 or
an alternative chemical treatment, such as Rubdium
Chloride is a mandatory step.24 Winnaker26 reported that
transformation efficiencies can reach up to 107 transformants
mg21 of DNA, which seems high but in reality this corresponds to only one molecule per 10 000 plasmid molecules
actually entering the cell.26 Thus, such finding is clearly
demonstrating that the CaCl2 protocol is relatively inefficient23 and since E. coli transformation is an essential step
in many cloning procedures, it is desirable to make it as efficient as possible.24
This study compares the CaCl2 method of transformation
to ultrasound treatment.
Ultrasound
Ultrasound is a well-established laboratory technique of cell
disruption27 seen to have considerable potential for use as a
transformant.18, 20, 28 – 30 The interest in the use of ultrasound as a means of transforming bacteria arises from observations that homogenous cell suspensions when exposed to
ultrasound exhibit cellular damage to varying extents.31
These effects range from apparently unaffected cells, to permeabilized cells, to fully lysed cells.32
Ultrasound has immediate advantages over its competing
technologies of both electroporation, which requires a low
ionic medium and high-voltage: and conjugation which
requires direct cell –cell contact. Unlike these techniques—
ultrasound does not have any stringent ionic media or
voltage requirements, making it an attractive alternative procedure.13 – 20 Ultrasound as a mechanical method is more versatile and less dependent on cell type.16
Cavitation (an effect generated by ultrasound) is considered
the major mechanism responsible for causing increased
membrane permeability, hence, the alterations seen among
biological tissues and possibly bacteria during transformation.11, 20, 30, 33 – 35 These changes are due to the stress
induced via ultrasound.36 Cavitation refers to the growth,
oscillation and collapse of microbubbles in an acoustic
field20, 33, 34 which induces the formation of pores in the
bacterial membrane that can permit transformation.35 The
pore sizes are 30– 100 nm, with the membrane recovery
period being quite rapid—a few seconds or at most a minute.33
Destruction of micro-organisms by ultrasound has been of
great interest37, 38 whereby the reduction in light emission
from a seawater suspension of rod shaped Photobacterium
fisheri has been recorded, following the use of ultrasound
(375 kHz).37 Scherba et al.39 also found that the relative percentage kill of bacteria, fungi and viruses in aqueous solutions,
increases with increased exposure and intensity of ultrasound.
Ultrasound can have two functions—it can allow for bacterial
transformation or cause bacterial cell death. Song et al.13 has
already demonstrated that ultrasound application is an efficient method for inducing transformation into bacteria,
however, the optimal conditions for E. coli had not been established. Thus, this study investigates how ultrasound exposure
affects the ability of E. coli HB101 to take up plasmid DNA.
Materials and methods
An overnight culture of E. coli HB101 (wild strain lacking plasmids) was prepared and used as recipients for 20 ml of pBR322
plasmid. The plasmid was prepared using the Wizard Plus SV
Minipreps DNA Purification System, and had a concentration
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of 19.5 mg ml21. To induce artificial competence, either CaCl2
or ultrasound was used. Two concentrations of CaCl2 were
investigated—0.05 mM (as previously used in a preliminary
experiment) and 50 mM (a concentration that is routinely
used in laboratory conditions)13, 23 and the heat shock protocol
was adopted.9, 23 This was designed to show whether a low and
a high concentration of CaCl2 could induce transformation in
E. coli, and how they compared in terms of transformation efficiency. Different ultrasound exposures were examined using a
48 kHz ultrasound bath at 228C13 ranging from 10 to 1200 s.
For each treatment, three sets of selective plates had been prepared [LB þ Ampicillin (AMP) antibiotic (50 mg ml21)] allowing for the positive selection of transformants. Results were
taken after 24 and 48 h of incubation of bacterial plates at
378C.
As a positive control, to establish the effect of ultrasound
on E. coli growth, 1026 dilutions of an overnight bacterial
culture (where the average viable number of bacteria ml21
of culture was 750 000 000) was prepared and subjected to
ultrasound exposures ranging from 0 to 1200 s.
Data analysis
The number of E. coli colonies and were counted and
averages taken. The transformation efficiency was calculated
and expressed as the number of transformants mg21 of DNA,
using the following formula:
Transformation Efficiency
¼
Number of E:coli colonies growing on LB/AMP plates
Amount of plasmid DNA utilized in experiment
The number of colonies were taken directly from plates.
The amount of plasmid DNA utilized was calculated using
the following formula: plasmid DNA (mg) ¼ concentration
of DNA (mg/ml) * volume of DNA (ml). The result of
which was 0.39 mg (0.0195 mg/ml*20 ml).
This calculation gives an indication of how effective the
transformation treatments were in getting plasmid DNA
into E. coli cells. The values give the number of E. coli
cells transformed by 1 mg of DNA.
Statistical analysis
Previous literature had not identified the type of relationship
between ultrasound exposure and transformation efficiency.
Therefore the data relating to the effect of ultrasound
exposure on transformation was analysed using R 2 values40
which had been calculated for each type of trend line
added (linear, exponential or logarithmic)41 using
Microsoft Excel. The R 2 values provide the square of the
Pearson product moment correlation coefficient and so
demonstrates the strength of the relationship, while the
trend lines establish the type of trend that best fit the data.
R 2 values, based on a logarithmic transformation of the
data gave the best values over the exponential and linear
trend line analyses, and it is the logarithmic analysis that is
therefore quoted in this paper.
T-tests were carried out using Microsoft Excel to see if
there was a significant difference between the two CaCl2
concentrations investigated after 24 and 48 h:
The formula for the t-test is as follows:42
Differences between the means
Standard error of differences between means
A comparison was made between CaCl2 and 10 s ultrasound exposure, using two tailed t-test analyses, based on
the data after 24 h.
Results
Two artificial transformation methods—ultrasound and
CaCl2 were compared. Using the 48 kHz ultrasound bath,
suspensions of E. coli broth culture and plasmid DNA,
were subjected to varying exposures of ultrasound. To investigate the effect of CaCl2, the heat shock protocol was used 9,
23
and two concentrations were investigated. Statistical
analysis of the results (t-tests) was conducted.
Ultrasound exposure does have an impact on E. coli transformation. A negative correlation was established between
ultrasound exposure and transformation efficiency, after 24
and 48 h of incubation (Fig. 1).
Ten seconds ultrasound exposure gave the best transformation efficiency values: 148.72 transformants mg21 of DNA
after 24 h and 674.36 transformants mg21 of DNA after
48 h of incubation. It should be recognized that this apparent
increase in transformation frequency after extended incubation could mean that the cells are being sub-lethally
injured by the treatment and therefore take longer to
recover and form colonies. 1200 s ultrasound exposure was
the least efficient: a transformation efficiency of 33.33 transformants mg21 of DNA was achieved after 24 h of incubation and 66.67 transformants mg21 of DNA after 48 h
(Fig. 1).
The R 2 values associated with the logarithmic analysis are
demonstrated in Fig. 1 and show the highly significant
relationship between ultrasound duration and transformation efficiency.
Both 0.05 and 50 mM CaCl2 can permit transformation in
E. coli HB101. The transformation efficiency was greater
using 50 mM CaCl2 than 0.05 mM CaCl2, both after 24
and 48 h of incubation (Table 1). Thus, CaCl2 does have
an effect on E. coli transformation.
The probability at the 95% confidence limit ¼ 0.05. A significant difference is achieved when the P-value of the t-test
is smaller than P ¼ 0.05. On comparison of 0.05 and 50 mM
CaCl2 after 24 and 48 h incubation, the P-values calculated
were smaller than the P ¼ 0.05 value, indicating a significant
difference between the two concentrations of CaCl2.
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Figure 1. The effect of different ultrasound exposures on E. coli HB101 transformation after a 24 and 48 h incubation period. Logarithmic trend lines and
R 2 values have been added.
Table 1. Effect of CaCl2 on E. coli HB101 transformation efficiency
Hours of incubation
Concentration CaCl2
Transformation efficiency/
Transformants mg21 of
plasmid DNA
................................................................................................................
24
48
0.05 mM
4.70
50 mM
118.80
P-value of t-test
2.20504E205
0.05 mM
10.26
50 mM
503.42
P-value of t-test
0.000675451
Table 2. T-test analyses comparing the different concentrations of
CaCl2 with 10 s exposure of ultrasound
Treatment
P-value of
t-test
Probability at 95%
confidence limit
Significant or
non-significant
0.005
0.05
Significant
0.074
0.05
Non-significant
................................................................................................................
10 s ultrasound
vs. 0.05 mM CaCl2
10 s ultrasound
vs. 50 mM CaCl2
Table 2 above shows the results of the t-tests, comparing
10 s ultrasound exposure and CaCl2 (0.05 and 50 mM)
after 24 h incubation.
Discussion
The use of ultrasound as a transformant is thought to be
simpler in relation to electroporation—the gold standard
laboratory technique adopted in genetic transformation.12,
Research by Song et al.13 has demonstrated that 10 s ultrasound exposure was more efficient than either electroporation or conjugation, during the transformation of P. putida
UWC1. This study, however, focused on the comparison
between ultrasound and CaCl2 induced transformation of
E. coli HB101. The positive control experiment showed
that ultrasound does have a detrimental effect on E. coli
growth over a time period of 1200 s (data shown in
Fig. 2). The data in Table 2 shows there was a significant
difference between the results obtained between the
0.05 mM CaCl2 and 10 s ultrasound exposure, and a nonsignificant difference between 50 mM CaCl2 and 10 s ultrasound exposure. These results indicate that 10 s ultrasound
exposure is more efficient in terms of transformation than
0.05 mM CaCl2; while 50 mM CaCl2 can yield transformation efficiencies similar to the efficiencies recorded at 10 s
ultrasound exposure. Hence, the 10 s ultrasound and
50 mM CaCl2 treatments for inducing artificial competence
are just as efficient at E. coli transformation.
CaCl2 induces artificial competence into bacterial cells,
encouraging transformation.1, 9, 13, 18, 23 – 25 It causes the
plasmid DNA to bind the surface of E. coli cells18, 23 and
when heat shock is applied (the rise in temperature to
428C)9, 23 the plasmid is incorporated into the cell.
Bacterial cell membranes are permeable to the chloride
ions and not to the calcium ions: so as the chloride enters
the cell, there is an influx of water molecules causing the bacterium to swell—a requirement necessary for DNA uptake.9
The CaCl2 concentration therefore, is an important consideration in transformation procedures.9 From the data obtained
investigating the effect of CaCl2 on E. coli transformation, it
can be deduced that CaCl2 is a positive factor promoting
16
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Figure 2. The negative association of ultrasound exposure on the average number of bacteria ml21 of culture.
transformation where 50 mM gave a higher transformation
efficiency than 0.05 mM (Table 1), reinforced by the t-tests
revealing significant relationships.
Figure 1 demonstrated a negative association between
ultrasound duration and transformation efficiency, with
the best efficiency being recorded at 10 s ultrasound
exposure and the least efficient at 1200 s exposure.
Although 10 s was recorded to give the best transformation
efficiency, there may be a critical period between 0 and 10 s
exposure that could have potentially resulted in an
increased efficiency. The results from this experiment and
the results from Song et al.13 have shown that 10 s ultrasound exposure is the most efficient in inducing DNA delivery from a plasmid source, into the Gram-negative
bacteria—E. coli HB101 and P. putida UWC1. This
reinforces the idea that ultrasound, as a mechanical
method is less dependent on cell types16 in comparison to
other transformation methods.
Ultrasound is a physical method, while CaCl2 is a chemical treatment, able to induce a state of competence.
Ultrasound is known to induce temporary porosity into bacterial cells to help facilitate the uptake of plasmid DNA,13
therefore it must be at 10 s exposure that the formation
and size of pores in the membrane to facilitate transformation, were the most optimal. The collapse of cavitation
bubbles generates the shock waves needed to cause localized
rupture to the membrane, leading to the uptake of exogenous
plasmid material.16
The expression of DNA in cells depends on the balance
between transient cell damage and cell death.16 This can be
explained by the effects of cavitation, which can induce
cell death or cell permeabilization without affecting cell viability.16, 20, 31, 32
Thus, at increased ultrasound exposures, the degree of
irreversible cell damage must be greater than the cell permeabilization15 leading to a lower transformation efficiency and
greater cell death. The 48 kHz ultrasound bath belongs to the
power ultrasound classification11 and operates at lowfrequency. This allowed cell permeabilization to occur at
low ultrasound exposure, and a greater amount of cell
death to occur at prolonged ultrasound exposures.
The R 2 values recorded were 0.8591 after 24 h and
0.7991 after 48 h (Fig. 1) which indicates the presence of a
direct relationship. It was demonstrated that after 48 h, the
transformation efficiency increased in comparison to the
efficiencies calculated after 24 h (Fig. 1). This is accounted
for by the difference in the initial bacterial cell numbers.
This also demonstrates the integrity of the plasmid,
since the new generation of cells produced would have
received the plasmid. Plasmid integrity is an important consideration when it comes to ultrasound exposure, since
research has demonstrated that 30 s exposure damages
plasmid DNA.13 Hence, this effect may possibly have
occurred at longer ultrasound exposures in this experiment.
However, gel electrophoresis could have been done to
confirm this effect, as adopted by Song et al.13
Conclusion
In order for ultrasound to be a valid alternative to currently
available methods of transformation, the transformation efficiency of ultrasound-mediated DNA delivery has to be
assessed with regards to the specific species under study.32
This study showed that ultrasound exposure and the concentration of CaCl2 does have an impact on E. coli HB101
transformation. Since E. coli is incapable of naturally undergoing transformation, artificial methods are required, like the
application of ultrasound or CaCl2. 10 s ultrasound exposure
was shown to be the most efficient for E. coli transformation,
and a negative association between ultrasound duration and
transformation efficiency was observed. Even at a low concentration of CaCl2 (0.05 mM) E. coli transformation was
shown to occur, however the presence of a higher concentration (50 mM) produced a greater number of
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transformants. A negative association between E. coli viability and ultrasound exposure was also identified. Literature
has demonstrated that cavitation is responsible for cellular
changes and the loss of viability in E. coli.32
Acknowledgements
I would like to thank my supervisor, Dr Ian Turner, for his
constant support and guidance throughout the completion
of this project: without which it would not have been possible. I would also like to thank Richard Duff, Dr Trevor
Neal and Rabinder Hayer for their valuable input into the
project.
Funding
This work was funded by the University of Derby.
Author biography
Kimran Hayer recently completed her Biology degree at the
University of Derby and obtained a first class honours,
together with the Institute of Biology (IOB) Award for best
student performance. It was her final year project that investigated the use of ultrasound as an alternative to bacterial
transformation, which stimulated her interest in the fields
of microbiology and genetics. Kimran is now at
Nottingham University pursuing her research interests and
working on a PhD project. The project looks at filamentous
fungal enzymes for the saccharification of biomass and is
likely to focus on understanding germination and hydrolase
expression in Aspergillus niger.
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