Note

A simple and rapid method of bacterial transformation

Gottfried Wilharm, Daniela Lepka, Franziska Faber, Julia Hofmann, Tobias Kerrinnes, Evelyn Skiebe
Robert Koch-Institute, Wernigerode Branch, Burgstr. 37, D-38855 Wernigerode, Germany
A b s t r a c t a r t i c l e i n f o
Article history:
Received 19 October 2009
Received in revised form 19 November 2009
Accepted 2 December 2009
Keywords:
Transformation
Nanopiercing
Yoshida effect
Sepiolite
E. coli
Acinetobacter
Yersinia
Recently, a unique method for bacterial transformation using nanobers to inoculate DNA has been
developed by Naoto Yoshida and colleagues. We have veried the principle, transforming Escherichia coli,
Yersinia enterocolitica and Acinetobacter baumannii, and have established a user-friendly protocol. A buffered
suspension of sepiolitean inexpensive, brous yet inoffensive mineralis mixed with bacteria and
transforming DNA and the mixture directly spread on selective agar.
2010 Elsevier B.V. All rights reserved.
Genetic engineering largely relies on the ability to introduce DNA
into the cells to be manipulated. The most widely used methods for
transformation of bacteria are chemical transformation and electro-
poration protocols (Yoshida and Sato, 2009). Since 2001, Yoshida and
colleagues have publisheda couple of exciting papers describing a novel
transformation method based on the inoculation of transforming DNA
into bacteria by means of mineral nanobers (Yoshida et al., 2001, 2002,
2007; Yoshida and Saeki, 2004; Yoshida and Takebe, 2006; Yoshida,
2007; Yoshida and Ide, 2008; Yoshida and Sato, 2009). This method,
termed Tribos transformation or hydrogel exposure method, is
remarkably simple. A colloidal solution containing mineral nanobers
is mixed with bacteria and transforming DNA and plated on selective
agar plates. The sliding frictionforces, arising between the surface of the
agar and the stir stick when bacteria are spread, result in penetration of
bacterial cells. This underlying phenomenon, termed Yoshida effect
(Yoshida, 2007), leads to inoculation of the transforming DNA which is
adsorbed to the mineral nanobers. Inside the cell, the DNA is probably
displaced by competition with small nucleic acids (Yoshida and Ide,
2008) so that it can be maintained and expressed (Fig. 1).
Strikingly, littlenoticehas beentakenof this veryinterestinginvention
as we found no citations referring to the method other then self-citations.
Possible reasons could be (i) that it seemed or proved unreliable, (ii) that
most of the published work referred to the use of chrysotile asbestos
bers (Yoshida et al., 2001, 2002, 2007; Yoshida and Saeki, 2004; Yoshida
and Takebe, 2006; Yoshida and Ide, 2008) which are known to cause
considerable health risks (Landrigan et al., 1999), or (iii) that in some of
their papers the authors suggest the usage of a specic apparatus for
optimized application of sliding friction forces (Yoshida, 2007; Yoshida
and Ide, 2008), all of which could discourage possible users.
Here, we have conrmed the principle and have optimized this
method for easy, cost-effective and harmless application.
First, due to the obvious health risks associated with chrysotile, we
decided to test the usefulness of sepiolite which has been mentioned
by Yoshida and colleagues as an alternative source of nanobers
(Yoshida and Sato, 2009). Sepiolite is a naturally occurring magne-
sium silicate and deposits are exploited mainly for the production of
absorbent such as pet litter. Due to its structure of very thin crystals it
is also used as asbestos substitute and the biological effects of sepiolite
bers have been shown to be mild compared with those of asbestos
(Koshi et al., 1991). According to Bellmann et al. (1997), Spanish
sepiolite has no carcinogenic potential and a lower biological activity
compared to sepiolite from Finland and China, likely due to minor
fractions of bers with a length above 5 m and due to the low
biodurability of Spanish sepiolite. Therefore, sepiolite mined in Spain
with a mean ber length of 2 m and a mean diameter of 20 nm was
purchased from Kremer Pigmente GmbH & Co. KG, Germany (cat. no.
58945) and used for our transformation studies.
Yoshida and Ide (2008) described a quite labor-intensive method
to receive a colloidal solution containing the proper bers including
several centrifugation and ltration steps. To simplify the procedure
as much as possible, an autoclaved 4% suspension of sepiolite in
deionized water was used as starting material without fractionation.
The suspension was diluted to 0.01% (100 g/ml) sepiolite in a sterile
solution of 5 mM HEPES pH 7.4, 200 mM KCl (sepiolite suspension).
From a logarithmic-phase culture of E. coli Top10 (OD
600nm
0.5), 2 ml
Journal of Microbiological Methods 80 (2010) 215216
Corresponding author. Tel.: +49 3943 679 282; fax: +49 3943 679 207.
E-mail addresses: wilharmg@rki.de (G. Wilharm), lepkad@rki.de (D. Lepka),
faberf@rki.de (F. Faber), hofmannj@rki.de (J. Hofmann), kerrinnest@rki.de
(T. Kerrinnes), skiebee@rki.de (E. Skiebe).
0167-7012/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.mimet.2009.12.002
Contents lists available at ScienceDirect
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j our nal homepage: www. el sevi er. com/ l ocat e/ j mi cmet h
were centrifuged and the bacterial pellet was resuspended in 500 l of
sepiolite suspension. Then, to 100 l of the mixture, 50 ng of plasmid
DNA (pUC19 (Yanisch-Perron et al., 1985), size 2.7 kbp; pWH1266
(Hunger et al., 1990), size 8.9 kbp) was added and plated on 2% agar
plates containing LB medium and selective antibiotics (50 g/ml
ampicillin and 20 g/ml tetracycline, respectively). Best results for
transformation of E. coli Top10 were achieved with the following
plating procedure: 100 l of transforming mixture consisting of
sepiolite suspension, bacteria and DNA were spread on a 2% agar
plate pre-dried under the biological safety cabinet for approx. 10 min.
The suspension was spread with polystyrene or glass stir sticks until
liquid completely soaked into the agar which is indicated by a marked
increase of frictional resistance, and spreading was then continued for
additional 30 s. The transformation efciency of E. coli Top10 was in
the range of 110
5
transformants per g of plasmid in the case of
pWH1266 (8.9 kbp) and 210
6
in the case of pUC19 (2.7 kbp). This is
comparable to the results of Yoshida and Ide (2008) who achieved
610
6
transformants per g of pUC19 plasmid using fractionated
chrysotile whisker and their specially designed apparatus to maintain
dened vertical reaction forces on the stir stick. Since in most labs
standard agar plates are used with 1% agar, for the sake of
simplication we tested the transformation efciency in relation to
the agar concentration and found the transformation efciency on 2%
agar plates to be only slightly superior to 1% agar plates (110
5
versus
410
4
transformants per g of pWH1266).
The transformationefciencyof Acinetobacter baumannii ATCC17978
was comparable to that of E. coli Top10 (110
5
transformants per g of
plasmid in the case of pWH1266). Yersinia enterocolitica WA-314 was
transformed with pACYC184 (Chang and Cohen, 1978) (4.2 kbp) at a
frequency of about 110
5
transformants per g of plasmid.
Taken together, the simplest protocol: Autoclave a suspension of
0.01%sepiolite indeionized water supplemented with 5 mMHEPES and
200 mM KCl (sepiolite suspension). Centrifuge 500 l of a bacterial
culture (0.51 OD), resuspend it in 100 l sepiolite suspension, add
50 ng of transformingDNAandspreadit ona 12%agar plate containing
the appropriate selective agents; spreading should be continued for
approximately 30 s after liquid has soaked into the agar which is
indicated by an increase in the frictional resistance. If there is need for
optimization, varying the following parameters is promising: growth
phase of the bacteria (better logarithmic than stationary), variation of
the ratio between sepiolite and DNA, and modulation of sliding friction
during plating (varying the concentration of agar between 2 and 4%,
length and strength of plating).
The only method of comparable simplicity we are aware of, the
protocol by Chung et al. (1989), a chemical protocol, seems to be
largely restricted to transform E. coli, while the advantage of the
nanopiercing method is its physical nature, so that a broad
applicability for subcloning procedures can be expected. In support
of that, applicability of the original method was reported also for the
Gram-positive Bacillus subtilis (Yoshida and Sato, 2009).
Acknowledgement
We would like to thank Guido Werner for providing plasmids and
strains.
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Fig 1. Principle and procedure of the nanopiercing method. (a) Transforming DNA
adsorbs to sepiolite bers. (b) Transforming DNA, sepiolite suspension and bacteria are
mixed and plated on a selective agar medium; sliding friction forces developing during
plating cause piercing of bacterial cells with sepiolite bers. (c) Inside the bacterial cell,
transforming DNA possibly is competitively displaced from sepiolite bers by cellular
nucleic acids (Yoshida and Ide, 2008).
216 G. Wilharm et al. / Journal of Microbiological Methods 80 (2010) 215216