ORIGINAL RESEARCH PAPER

Surface-functionalized gold nanoparticles mediate bacterialtransformation: a nanobiotechnological approach

Saptarshi Chatterjee • Keka Sarkar

Received: 5 June 2013 / Accepted: 16 September 2013 / Published online: 8 October 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Transformation of bacteria is an important

step in molecular biology. Viral and non-virus-based

gene delivery techniques, including chemical/biolog-

ical and physical approaches, have been applied to

bacterial, mammalian and plant cells. E. coli is not

competent to take up DNA; hence, different methods

are used to incorporate plasmid DNA. A novel method

has been developed using glutathione-functionalized

gold nanoparticles to mediate transformation of plas-

mid DNA (pUC19) into E. coli DH5a that does not

require the preparation of competent cells. The

glutathione-functionalized gold nanoparticles acted

as a vector and facilitated the entry of DNA into the

host cell. The method also gave a higher transforma-

tion efficiency (4.2 9 107/lg DNA) compared to

2.3 9 105/lg DNA using the conventional CaCl2-

mediated method. It was also non-toxic to the

bacterium making it suitable for biotechnological

applications.

Keywords Cytotoxicity � Glutathione-

functionalized gold nanoparticles �Nanobiotechnology � Nanoparticles �Transformation

Introduction

Nanotechnology has evolved as a major interdisci-

plinary science. It has been explored for various

biological processes (Pinto-Alphandary et al. 2000;

Chan et al. 1998). Nanoparticles are typically smaller

compared to large biological molecules, such as

enzymes, receptors and antibodies, and thus offer

unprecedented interactions with biomolecules both on

the surface and inside the cell (Cai et al. 2008). The

well-studied nanoparticles include quantum dots,

carbon nanotubes and paramagnetic nanoparticles.

However, the gold nanoparticles remain a major

choice in case of biomedical applications because of

their unique optical properties, surface plasmon res-

onance and biocompatibility.

DNA transformation is important in all aspects of

molecular biology. It occurs naturally in some bacteria,

such as Micrococcus, Haemophilus and Bacillus (Mi-

chod et al. 1988), but it can also be affected by artificial

means in other cells. Such cells that are capable of being

transformed are termed as competent cells. E. coli

DH5a that is not naturally competent can be made

competent by using a CaCl2 treatment and subsequent

thermal shock (Cohen et al. 1972). Although several

Electronic supplementary material The online version ofthis article (doi:10.1007/s10529-013-1360-x) contains supple-mentary material, which is available to authorized users.

S. Chatterjee � K. Sarkar (&)

Department of Microbiology, University of Kalyani,

Calcutta, West Bengal, India

e-mail: keka.micro@gmail.com

S. Chatterjee

e-mail: saptarshi_gcc07@yahoo.co.in

123

Biotechnol Lett (2014) 36:265–271

DOI 10.1007/s10529-013-1360-x

techniques have been tried to improve competence

(Prakash et al. 2011; Lederberg and Cohen 1974), the

technique of E. coli transformation remains highly

inefficient even using competent cells. Recent

approaches have used gold nanoparticle-mediated gene

delivery in animals (Kim et al. 2012) and plants. But no

such attempt has been made to use gold nanoparticle-

mediated delivery of plasmid DNA in bacteria.

The goal of the present study was to synthesize

glutathione-surface-functionalized gold nanoparticles,

characterize them and apply them for the transfer of pUC

19 gene into non-competent E. coli DH5a. Successful

transformation was indicated by the growth of ampicil-

lin-resistant colonies in medium containing the antibi-

otic. The transformation efficiency of the method was

evaluated along with comparison with a conventional

method. Finally, the biocompatibility of the synthesized

nanoparticle was also checked on E. coli DH5a.

Materials and methods

Preparation of glutathione-functionalized gold

nanoparticles

80 ml of 1 mM HAuCl4 in methanol and 40 ml 3 mM

glutathione were mixed and generated a cloudy white

suspension. It was reduced using 10 ml 10 mM

NaBH4 under constant stirring, which resulted in dark

brown suspension (Schaaff and Whetten 2000). This

was dried at 43 �C and then washed with methanol 2–3

times. Finally, the glutathione-functionalized gold

nanoparticles (Glu@Au) were filtered using 0.1 lm

filter and re-suspended in 25 ml nano-pure water.

Characterization of glutathione-functionalized

gold nanoparticles

Characterization of glutathione-functionalized gold

nanoparticle was conducted using spectrophotome-

tery, transmission electron microscopy, scanning

electron microscopy, dynamic light scattering, and

X-ray diffraction.

Transformation of non-competent E. coli

E. coli DH5a was used because it is ampicillin-sensitive,

whereas pUC19 plasmid was selected because it possesses

a ampicillin-resistant gene. Glutathione-functionalized

gold nanoparticles (30–70 lg/ml) were mixed with 1 ll

pUC19 DNA (from a stock of 1 ng/ll) for 2 h at 37 �C.

Subsequently, E. coli DH5a was grown on LB broth to an

OD600 value of*0.1 (i.e.*5 9 107 cells/ml). One ml of

the bacterial culture was centrifuged at 6,0009g for

1 min, and 20 ll pUC 19-Au nanoparticle mixture was

added to the pellet followed by 980 ll fresh LB medium,

mixed and incubated at 37 �C for 7 h with shaking. After

3 h, 100 ll stock culture was withdrawn hourly and plated

on LB agar medium containing 100 lg of ampicillin/ml

media. Plates were incubated at 37 �C for 24 h and the

c.f.u. value was calculated to evaluate the transformation

efficiency. The procedure is shown in Fig. 1 and was

carried out in triplicate.

Optimization of procedure and comparison

with conventional method

The amount of nanoparticle (30–70 lg/ml) added to

10 ll pUC 19 DNA and the incubation time for

Glu@Au-DNA with E.coli DH5a were optimized.

The efficiency of the glutathione-functionalized gold

nanoparticle-mediated transformation of non-compe-

tent E. coli DH5a was compared to the standard

CaCl2-mediated method. The plasmid of the trans-

formed bacterial cells was also isolated and compared

to commercially available plasmid.

Nanotoxicity study

The biocompatibility of the synthesized glutathione-

functionalized gold nanoparticle on E. coli DH5a was

checked from its growth curve (Chatterjee et al. 2011).

E.coli was grown with shaking in 50 ml LB broth

medium at 37 �C for 14 h. One ml was then transferred

to 100 ml LB broth in 250 ml conical flasks. Nano-

particles (25–100 lg/ml) were added into each flask. A

control without nanoparticle addition was also used.

Cultures were grown at 37 �C with shaking and the

growth rate was determined from the OD600 values.

Results

Characterization of glutathione-functionalized

gold nanoparticles

From transmission electron microscopy, the size of the

glutathione-functionalized gold nanoparticles was

266 Biotechnol Lett (2014) 36:265–271

123

2 nm and this was confirmed by SEM analysis

(Fig. 2). This also indicated the synthesized nanopar-

ticles were spherical. The Dynamic light scattering

indicated monodispersity (the degree of polydispersity

is 0.182) (Fig. 2). X-Ray diffraction (XRD) of the

dried gold nanoparticle powders is also shown in

Fig. 2. From spectrophotometry the plasmon reso-

nance peak of glutathione-functionalized gold nano-

particle was at 535 nm (Fig. 2). Though a shift of

1 nm in the localized surface plasmon resonance

(LSPR) was evident at 1 h of incubation, a decrease of

overall spectrum, as well as a 3 nm LSPR shift, was

seen on 5 h of incubation. This indicated the stability

and interaction of Glu@Au nanoparticle-plasmid

DNA complex.

Glu@Au nanoparticle-mediated bacterial

transformation

The ampicillin-resistant gene present in pUC19 was

transferred to E. coli DH5a using glutathione-

functionalized gold nanoparticles. The transformed

bacteria now grow in antibiotic-containing medium.

The transformation efficiency was 4.2 9 107/lg DNA

and was calculated using the formula:

Transformation efficiency ¼ number of transformedðcolonies=amount of DNAÞ

Since glutathione has an electrostatic interaction

with both gold nanoparticle and DNA, the gold

Fig. 1 Procedure of the glutathione-functionalized gold nanoparticle-mediated bacterial transformation shown schematically

Biotechnol Lett (2014) 36:265–271 267

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nanoparticles were surface-modified using glutathione

followed by interaction with plasmid DNA. The

carboxyl group (COO-) of the glycine residue elec-

trostatically interacts with the positively charged gold

nanoparticle to form glutathione-functionalized gold

nanoparticle. The free end (c-glutamine residue) of

glutathione now possesses an amine group and a

carboxyl group. Of the two, the amine group non-

specifically interacts with the negatively-charged

phosphate group of DNA forming a reversible elec-

trostatic complex of gold-glutathione-DNA. Due to

ionic variation, this complex cleaves within the

bacterial cell, liberating the intact plasmid DNA from

gold–glutathione complex.

Optimization of procedure and comparison

with conventional method

The optimal concentration of glutathione-functiona-

lized gold nanoparticles to react with 1 ll pUC19

DNA (from a stock of 1 ng/ll) was 50 lg/ml (Sup-

plementary Fig. 1). Lower concentrations diminished

the efficiency of transformation whereas higher con-

centrations did not significantly improve transforma-

tion. The optimum time for the interaction of the nano-

plasmid complex(Glu@Au-pUC19) with E. coli

DH5a was 5 h (Supplementary Fig. 2).

The process was compared to theconventional CaCl2-

mediated transformation in terms of transformation

Fig. 2 a TEM image of the

glutathione-functionalized

gold nanoparticle

(Glu@Au) of 2 nm. b SEM

image of spherical

glutathione-functionalized

gold nanoparticle of 2 nm.

c DLS data of the Glu@Au

nanoparticle showing the

degree of dispersity (the

degree of polydispersity is

0.182). d XRD of the gold

nanoparticle powders

performed within a 2h range

of 20�–80� using Cu K aradiation which has

diffraction peaks at 2h(38.2�, 44.4�, 64.6�, and

77.5�), that can be indexed

to (111), (200), (220) and

(311) planes of gold in the

cubic phase. e Spectroscopy

of the glutathione-

functionalized gold

nanoparticle (Glu@Au)-

pUC19 interaction at 1 h

(a) and 5 h (b) of incubation

showing changes of surface

plasmon resonance (SPR)

peak and visual

agglomeration

268 Biotechnol Lett (2014) 36:265–271

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efficiency. The transformation efficiency for the glu-

tathione-functionalized gold nanoparticle-mediated

transformation was 4.2 9 107/lg DNA compared to

that 2.3 9 105/lg DNA using the conventional CaCl2-

mediated transformation. This result clearly shows an

advantage of our process over the conventional method.

Finally, the isolation of plasmid DNA from the trans-

formed E. coli gave indirect evidence of successful

transformation (Fig. 3). Thus, this study shows a bio-

application of surface-functionalized gold nanoparticle.

Further research is required to make this transformation

process generalized for other organisms.

Nanotoxicity study

The growth of E.coli DH5a with various concentra-

tions of Glu@Au nanoparticles (25–100 lg/ml)

showed no cytotoxicity (Fig. 4). There were no

difference in the growth of untreated and nanoparti-

cle-treated bacteria. This indicated that the nanopar-

ticles are non-toxic to E.coli DH5a. After 8 h of

incubation. the dry weight of untreated bacteria after

was 0.72 ± 0.012 g/l that remained unaltered for

Fig. 2 continued

Fig. 3 Agarose gel electrophoresis of the extracted plasmid

from transformed E. coli DH5a (Lane 1) and comparison with

commercially available pUC19 plasmid (Lane 2) indicating

successful transformation

Biotechnol Lett (2014) 36:265–271 269

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Glu@Au nanoparticle-treated bacteria. Thus the syn-

thesized glutathione-functionalized gold nanoparticles

were suitable for biological applications.

Discussion

Transformation of bacteria is important in molecular

biology. However, a generalized method for transfor-

mation of different bacteria is unavailable. Several

attempts are made to transform various bacteria (Wirth

et al. 1989) with plasmids and to obtain a high

efficiency (Inoue et al. 1990).

Here, we have synthesized gold nanoparticles of

2 nm that were surface functionalized using glutathi-

one. The characterization of the nanoparticle was done

by TEM, SEM, DLS and XRD. Finally, we developed

a novel method of bacterial transformation using

glutathione-functionalized gold nanoparticles that

gave a higher transformation efficiency. This method

has the potential to transform bacteria that are

difficult-to-transform.

Non-viral delivery of gene is an important step of

cloning and routinely used in molecular biology.

Initially, emphasis was given on the method of

preparation, storage and optimization of competent

cells (Tang et al. 1994; Nishimura et al. 1990). Later,

various technologies were used in the non-viral gene

delivery methods (Song et al. 2007) to bring higher

transformation efficiencies than the natural and con-

ventional methods.

Although there are several reports of the application

of gold nanoparticles in the targeted delivery of genes

in animal cell lines (Sandhu et al. 2002), no such

attempts have been made to transform bacteria using

surface-functionalized gold nanoparticles. This

method is simple, possesses high efficiency and also

does not require preparation of competent cells. The

process was also optimized to obtain highest transfor-

mation efficiency. This method can be compared to

other methods available for increasing the transforma-

tion efficiency of bacteria (Supplementary Table 1).

The viability of bacteria and integrity of plasmid

are two important factors in the transformation of

Fig. 4 Growth curve of

E. coli DH5a with Glu@Au

nanoparticles (25–100 lg/

ml) indicating

biocompatibility of the

synthesized glutathione-

functionalized nanoparticles

270 Biotechnol Lett (2014) 36:265–271

123

bacteria. Hence, the biocompatibility of the synthe-

sized glutathione-functionalized gold nanoparticle on

E. coli DH5a was studied. Although there are reports

of toxicity caused by cationic and anionic side-chains-

functionalized gold nanoparticles (Goodman et al.

2004), the synthesized nanoparticles showed no cyto-

toxicity. The isolation of plasmid DNA from the

transformed E. coli showed the integrity of the

plasmid.

However, further research is required to generalize

this method and to find out the molecular mechanism.

The role of particle size and shape that depends on the

method of synthesis lies out of the scope of our

research. It may play an important role in the uptake of

the nanoparticle as shown in similar study on mam-

malian cells (Chithrani et al. 2006). In conclusion, a

nanobiotechnological approach has been taken to

develop a novel method for the transformation of

bacteria using glutathione-functionalized gold nano-

particles that is simple and have higher efficiency.

Acknowledgments This research work has been carried out

with the financial support of Department of Science &

Technology, Government of India (Project-Nanomission: SR/

NM/NS-48/2009) and University of Kalyani, Nadia, West

Bengal.

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