RESEARCH COMMUNICATIONS

Heat shock at 37°C with plasmid cells followed by selection of the transformants in
medium supplemented with the respective antibiotics.
ligated at 37°C yields more number of The usual understanding pertaining to heat shock-
Escherichia coli transformants than mediated transformation event at 42°C is that sudden
plasmid ligated at 16°C: a possible role shift in temperature from 0°C (ice temperature) to 42°C
causes transient pore formation in the host cell (e.g. E.
of ligated plasmid conformation during coli) membrane, which in turn allows the ligated plasmid
heat shock to enter the cell. But whether this temperature shift also
affects the conformation of the ligated plasmid entering
Anjan Barman1, Praveen Kumar1,2, the host cell is not clearly understood. Generally, after
ligation at 16°C, the product is also subjected to a tem-
Vinay Kumar1,3, Robin Doley1 and
perature shift when heat shock is given along with the
Suvendra Kumar Ray1,* competent host cell at 42°C. This shift of about 26°C (i.e.
1
Department of Molecular Biology and Biotechnology, 42°C – 16°C = 26°C) is also likely to alter the plasmid
Tezpur University, Tezpur 784 028, India
2
Present address: Institute of Microbial Technology,
conformation, as it is known that temperature affects
Sector 39A, Chandigarh 160 036, India plasmid conformation5,6. Hence our hypothesis is that,
3
Present address: National Institute of Plant Genome Research, during transformation by heat shock, temperature shift-
Aruna Asaf Ali Marg, P.O. Box No. 10531, New Delhi 110 067, India induced altered conformation of the ligated plasmid
might have a role in the transformation event. A hypo-
The phenomenon of temperature effects on plasmid thetical model in support of the hypothesis is shown in
conformation has been well elucidated. However, the
Figure 1. Inside the bacterium, chromosomal DNA is
impact of change in plasmid conformation caused by
temperature shift (during the heat shock mode) on shown as a relaxed circle, whereas the plasmid is shown
transformation outcomes has not been reported till in a supercoiled state. This plasmid after isolation is
now. Here, we analyse transformation efficacy at 37°C transformation-efficient. Its efficiency can be attributed
of plasmid pBSKS ligated at three different tempera- to the natural conformation of the plasmid, which is nega-
tures (4°C, 16°C and 37°C) to Escherichia coli DH5α tively supercoiled. After restriction digestion the plasmid
strain. Though ligation was effective at all the three gets linearized, which is inefficient for transformation.
temperatures mentioned above, the number of trans- The linearized plasmid is ligated at 16°C, which attains a
formants was more with plasmid DNA ligated at 37°C relaxed conformation at this temperature. The transforma-
than at 4°C and 16°C. Our study indicates a possible tion efficiency of a relaxed plasmid might be low due to
role of plasmid conformation on the effect of heat
shock transformation.

Keywords: Escherichia coli, heat shock, ligation,

plasmid conformation, transformation.

TRANSFORMATION is an important and primordial approach

to isolate recombinants using horizontal gene transfer.
Bacterium Escherichia coli is regarded as naturally in-
competent for transformation1, though there are reports of
natural plasmid transformation in stationary phase E. coli
cells2 as well as for E. coli cells in environments with low
concentration of Ca++ ions3. Artificial competent cells of
E. coli are developed either for heat shock transformation
or for electroporation using specific protocols. Heat
shock is a simple and cost-effective method unlike elec-
troporation which requires sophisticated instrument such
as an electroporator, specific cuvettes and dialysis mem-
brane. Therefore, heat shock transformation in E. coli that Figure 1. The proposed effect of temperature on plasmid conforma-
was invented in 1970s is still a popular laboratory prac- tion during the heat shock transformation. A hypothetical model de-
tice among researchers1. scribing different conformations of a plasmid starting from isolation to
transformation. The plasmid isolated from a cell is naturally negatively
During heat shock transformation the steps followed supercoiled. After treatment with restriction endonuclease enzymes in
are4: (i) setting ligation at 16°C, (ii) exposure to heat vitro, the plasmid gets linearized and the torsion strain is removed. By
shock at 42°C for 90 sec and (iii) growth of the bacterial treating with ligase, the plasmid is ligated at 16°C to form a covalently
closed circular plasmid, which is in a relaxed state at that temperature.
During heat shock, the covalently closed circular plasmid attains posi-
*For correspondence. (e-mail: [email protected]) tively supercoiled state due to increase in temperature.

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its higher surface area as is evident from the slow migra- linearized plasmids were subjected to ligation at three
tion rate during electrophoresis. The plasmid when different temperatures, i.e. 4°, 16° and 37°C in order to
subjected to heat shock at 42°C might attain positive monitor success of the ligation process at all the three
supercoiled conformation due to temperature shift which temperatures. During the ligation set-up, ligation master-
might in turn enhance transformation efficiency. mix was prepared initially followed by addition of the
To prove our hypothesis, we have tried to explore the ligase enzyme at the end. To maintain equal concentra-
transformation efficiency with temperature shifts (described tion of the linearized plasmid DNA, the ligation-mixture
in Figure 2). Plasmids were ligated at 4°, 16° and 37°C was distributed equally into three tubes. All the reactions
and transformed at 37°C. This comparative study of heat were performed in ice to avoid ligase activity prior to
shock-mediated transformation at 37°C with products their transfer to respective temperature conditions.
ligated at varied temperatures demonstrates that plasmid The ligation mixture kept at different temperatures was
conformation might play a role during uptake of plasmid subjected to electrophoresis in two ways. One with EtBr
by competent cells. incorporated into the gel prior to electrophoresis and the
Plasmids were isolated from E. coli DH5α cells harbour- other with the gel stained after electrophoresis. Multiple
ing pBSKS. The competent E. coli DH5α bacterial cells bands were observed in the gel after ligation, suggesting
used for the experiment were prepared in the laboratory. that ligation was successful at all the three temperatures
E. coli DH5α strains harbouring pBSKS were grown in (Figure 3). As a band was observed below 3.0 kb (size of
Luria–Bertani (LB) broth (Himedia, Mumbai, 20 g/l) pBSKS is 3.0 kb) in both the gels, we predicted that
incubated at 37°C and 200 rpm in an orbital shaker this might be the supercoiled plasmid resulting from in-
incubator (Orbitek, Syngenics Biotech, SN-2007400128) tramolecular ligation. The intensity of the bands in the
for about 16 h. For plating purposes, 1.5% agar (Himedia, gel in Figure 3 b is more than that of the gel in Figure 3 a
Mumbai) was added to the LB broth and solidified. because the gel in Figure 3 b was stained later with EtBr.
pBSKS plasmids were isolated from cultured E. coli The linearized pBSKS in both the gels (Figure 3) was
DH5α strains harbouring pBSKS using commercial plas- running as a 3.0 kb band, whereas the supercoiled DNA
mid isolation kit (AxyGen, USA). The quality of the iso- in gel containing EtBr (Figure 3 a) is moving as ~ 1.7 kb
lated plasmid was checked by agarose gel electrophoresis band and the supercoiled DNA in the gel without EtBr
(0.8% agarose; SRL, Mumbai) using ethidium bromide (Figure 3 b) is moving like a ~ 2.2 kb band. In the former
(EtBr; 0.5 μg/ml conc., Biorad, Mumbai) at 70 V, case the plasmid DNA has undergone more supercoiling
250 mA, 1 h. due to intercalation of EtBr. There is a little difference in
pBSKS plasmids isolated from cultured E. coli DH5α the migration patterns among the supercoiled DNA
cells were digested with EcoRI (Fermentas, USA, 5U/μl ligated at different temperatures (Figure 3 b). The super-
stock) following the manufacturer’s instructions. Restric- coiled plasmid obtained during ligation at 37°C is moving
tion digestion of plasmid was checked by agarose gel faster than that obtained at 16°C and 4°C. In conclusion,
electrophoresis (0.8% agarose, 70 V, 250 mA, 1 h). in this experiment we demonstrate that ligation of
Digested product was then purified using plasmid purifi- linearized pBSKS had occurred well at 4°C and 37°C
cation kit (Qiagen, Valencia, CA, USA). Gel-extraction apart from at the usual temperatures of 16°C. We had
procedure to isolate digested DNA was avoided com- also demonstrated earlier7 that λDNA HindIII fragments
pletely to keep away any effect of EtBr on plasmid DNA were ligated efficiently when subjected to ligation at
conformation. Digested pBSKS was then kept for ligation 37°C and 16°C.
reaction with T4 DNA ligase (Fermentas, USA, 5U/μl To examine the transformation efficacy of ligated
stock) at 4°, 16° and 37°C separately for 12 h. Ligation pBSKS at 4°, 16° and 37°C, the ligated plasmids were
was checked by 0.8% agarose gel electrophoresis in two transformed to competent E. coli DH5α cells by heat
ways – (a) with EtBr (0.5 μg/ml conc.) incorporated into shock at 37°C separately. From earlier perceptions,
the gel prior to electrophoresis and (b) staining the gel higher temperature (as 37°C is used for heat shock in our
with EtBr (0.5 μg/ml conc.) post-electrophoresis; other case) is thought to introduce positive supercoils in plas-
conditions were identical for both the gels. mid ligated at lower temperatures (as 4° and 16°C have
Competent E. coli DH5α cells were prepared following been used in our experiment). Whereas heat shock at
CaCl2 treatment method4. Transformation of the compe- higher temperatures like 37°C will not introduce positive
tent cells was performed by heat shock at 37°C for 90 sec supercoils in plasmid molecules ligated at 37°C. So, dur-
with the ligated plasmids obtained from all the three ing transformation, heat shock at 37°C with ligation
ligation set-up events, i.e. 4°, 16° and 37°C separately. products at lower temperatures such as 4°C and 16°C is
Transformants were then checked by plating the cells on expected to yield better transformation outcomes than
LB-agar plates containing antibiotic ampicillin (50 μg/ml the ligated product at higher temperatures like 37°C
conc.) incubated at 37°C for the requisite period of time. (Figure 1).
In the present set of experiments, isolated pBSKS plas- Heat shock-mediated transformation (at 37°C) of E.
mids were first linearized by digesting with EcoRI. The coli DH5α, set separately with intact pBSKS, digested
748 CURRENT SCIENCE, VOL. 104, NO. 6, 25 MARCH 2013
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pBSKS, and ligated pBSKS from ligation tubes kept at performed at 4°C and 16°C. In fact, ligation performed at
4°, 16° and 37°C respectively, manifested interesting out- 4°C resulted in more number of transformants than at
comes. Figure 4 depicts a comparison of the transforma- 16°C. The consequence is surprising because ligation is
tion events set at the respective experimental conditions. usually performed at 16°C and our finding shows that the
Transformation with intact pBSKS yielded maximum number of transformants with products ligated at 37°C
average transformants per plate. This might be due to the and 4°C was more than that at 16°C. The higher number
integral conformation of plasmid, as these were not treated of transformants of plasmids ligated at 37°C and 4°C
with EcoRI. Digested or linearized pBSKS showed negli- might be due to the different levels of supercoiled state of
gible transformant yield. Among the ligated products the ligated plasmid during heat shock at 37°C. However,
from three separate ligation events, transformation with transformation by electroporation with the same ligated
pBSKS ligated at 37°C resulted in highest average trans- samples might reveal a different story.
formants per plate. The next best transformation yield The observation of slow migration of the DNA bands
obtained was with pBSKS ligated at 4°C followed by at lower front without EtBr suggests that the presence of
16°C. The average transformants per plate resulting with EtBr in the gel introduces supercoils in the plasmid. Even
pBSKS ligated at three temperatures is in descending without EtBr the ligated plasmid is able to attain some
order as 107.75 > 82.75 > 58, i.e. 37° > 4° > 16°C. The amount of supercoiling. The incidence of supercoiling
transformation event was executed at least three times to here is likely to be due to the electrophoresis conditions
obtain the best result and all the events showed similar as well as temperature of buffers used for treatments.
outcomes. Figure 4 is a graphical representation of the This implies that the temperature has a role in alteration
average transformants per plate with respect to ligation of conformation in covalently closed circular DNA.
conditions. The possible explanation for this is discussed The surprising inquiry in this study is pertaining to the
below. low transformant yield with 16°C ligation product as
In this study we have shown that linearized pBSKS against the other two. Then, what might be the reason be-
plasmid ligated at 37°C yields more number of transfor- hind the high transformant yield with plasmid DNA
mants by heat shock at 37°C than when ligation was ligated at 37°C? There is least possibility of increment in
the intramolecular ligation happening during ligation at
37°C as manifested by the similar intensity of the super-
coiled plasmid bands in the gel. In fact the 37°C ligated
product showed lower intensity compared to the other
two. Hence, increased number of intra-molecular liga-
tions leading to more number of transformant yields dur-
ing transformation would not hold. A critical observation
of the fact is necessary to arrive at a conclusion. Earlier,
transformation of E. coli at 37°C has been shown in food-
stuffs8. Recently, transformation viability in E. coli with
plasmid pUC19 by heat shock at 37°C and three other
temperatures has also been reported9. Adding to this, a
group reported about a finding pertaining to transforma-
tion feasibility of pre-incubated plate at 37°C that
resulted in double time the transformation efficiency in
comparison with the routine 42°C heat shock treatment10.
Considering their report, we chose 37°C temperature to
be feasible for transformation by heat shock as well.
However, in none of the previous studies involving trans-
formation at 37°C has any comparison been made for
Figure 2. Schematic representation of the plasmid conformations
proposed during the course of experiments designed in this study. 1, transformation efficiency of products ligated at different
The isolated pBSKS (negatively supercoiled) was treated with EcoRI to temperatures, which we have illustrated here.
obtain linearized pBSKS. 2, The linearized pBSKS was treated with T4 One of the rationalizations relating to transformation
DNA ligase for ligation followed by incubation at three different tem-
peratures separately at 4°C (2a), 16°C (2b) and 37°C (2c). Ligation re- by heat shock can be that DNA remains naturally in nega-
sults in covalently closed circular plasmids that are in relaxed tively supercoiled state inside the cell. So, negative
conformation at the respective temperatures. 3, Before heat shock, the supercoiled state of a plasmid can be attained easily from
ligated product is mixed with competent cell and incubated in ice
(~ 4°C). The lowering in temperature results 16°C (2b) and 37°C (2c) a relaxed state of the plasmid than that from a positive
ligated plasmid attaining negative supercoiled state, whereas the 4°C supercoiled state. In addition, the transient pore formed
ligated plasmid remains relaxed. 4, During heat shock at 37°C, the 4°C due to heat shock at 37°C is good enough for the uptake
(2a) and 16°C (2b) ligated plasmids attain positive supercoiled state
owing to increase in temperature, whereas the 37°C (2c) ligated prod- of both relaxed and less supercoiled (ligated at 37°C) as
uct attains relaxed conformation. well as the positive supercoiled form (ligated at 16°C and
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Figure 3. Agarose gels showing ligation of pBSKS plasmid at 4°, 16° and 37°C. a, Migration pattern of the ligated pBSKS plasmids at the
respective incubation temperatures on agarose gel, in which ethidium bromide (EtBr) was incorporated prior to electrophoresis. Lane 1, 1 kb DNA
ladder (Gene ruler, Fermentas, USA); Lanes 2, 4 and 6, Migration pattern of the ligated pBSKS plasmid at 4°, 16° and 37°C respectively. In all the
lanes equal amount of DNA has been loaded. Ligated plasmids are running as large-sized bands towards the upper end of the gel and also as bands
between 1.5 and 2.0 kb linear DNA. The former class belongs to the cocatemeric DNA moving slowest due to larger size and the latter class be-
longs to the supercoiled plasmid DNA which is running faster than the linear control DNA in lane 8. pBSKS ligated at 4°C loaded in lane 2 is mi-
grating faster than the ligated plasmids at 16° and 37°C, loaded in lanes 4 and 6 respectively. In fact, pBSKS ligated at 37°C is running the slowest
followed by plasmid ligated at 16°C, in this case; lane 8, The digested pBSKS plasmid as control. b, Migration pattern of the ligated pBSKS plas-
mids at the respective incubation temperatures on agarose gel, stained with EtBr post-electrophoresis. Lane 1, 1 kb DNA ladder; lanes 2, 4 and 6,
Migration pattern of the ligated pBSKS plasmid at 4°, 16° and 37°C respectively. In all the lanes equal amount of DNA has been loaded. Ligated
plasmids are running as large-sized bands towards the upper end of the gel, few in the middle and the lowermost bands are running as supercoiled
plasmids corresponding to 2.0–2.5 kb linear DNA ladder and are moving faster than the linearized control DNA in lane 8. The former class at the
topmost front of the gel is concatemeric DNA running slowest of all due their larger size. pBSKS ligated at 37°C loaded in lane 6 is migrating
faster than the ligated plasmids at 4°C and 16°C, loaded in the lanes 2 and 4 respectively. In fact, pBSKS ligated at 4°C is running the slowest fol-
lowed by ligated plasmid at 16°C, in this case; lane 8, Digested pBSKS plasmid (control). The experiment was performed thrice and the best of
three results is shown here.

Figure 4. A histogram depicting the average transformants per plate obtained. In the histogram, horizontal axis depicts the state of pBSKS used
for heat shock transformation such as temperature conditions kept as 4°, 16° and 37°C for ligation, and also the uncut and EcoRI cut plasmid. Ver-
tical axis shows the average number of transformants per plate obtained after the transformation event. Transformation with the intact undigested
plasmid yields the maximum average transformants per plate as shown by its highest stature followed by plasmid ligated at 37°, 4° and 16°C
respectively. This result is the best of the three sets of experiments carried out.

4°C). Apart from this, the role of plasmid conformation possible that the 16°C ligated product, which is positively
in expression of the antibiotic resistance gene cannot be supercoiled, experiences hindrance in multiplication and
ignored while drawing our conclusion about higher trans- gene expression after entering into the host bacterium.
formation efficiency of 37°C ligated product. It is known Higher number of transformants obtained in our experi-
that the supercoiled state of the plasmid influences gene ment with the 37°C ligated products was due to the high
expression in bacteria11,12. As the natural negative super- level of expression of the antibiotic resistant gene on the
coiled state of the plasmid is close to the relaxed state antibiotic supplemented medium (ampicillin in our case).
than the positive supercoiled state, the 37°C ligated plas- In this connection, it is also important to note that a
mid may be able to multiply and express well after enter- plasmid tends to attain the relaxed conformation from the
ing into the bacterium. On the other hand, it may be supercoiled conformation as the cells approach the sta-
750 CURRENT SCIENCE, VOL. 104, NO. 6, 25 MARCH 2013
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tionery phase from the exponential phase13. The above tions (e.g. linking number is an integer, the effect of salt
arguments regarding the difference in the antibiotic gene concentration and temperature on supercoiling, etc.) for
expression cannot support the observation of higher maintaining a covalently closed plasmid in relaxed state
transformants with 37°C ligated products, because the is most likely creating a hurdle for experimentation and
4°C ligated products also yielded higher transformants in critical analysis in this regard. In conclusion our study
comparison with the 16°C ligated products. The 4°C liga- has opened up new challenges for exploring the mecha-
tion products are more positively supercoiled than the nism behind the phenomenon of plasmid transformation
16°C products when shifted to 37°C. Therefore, explana- during heat shock, which is not clearly understood.
tion on the basis of the assumption of the difference in
the antibiotic resistant gene expression by different states
1. Cohen, S. N., Chang, A. C. Y. and Hsu, L., Nonchromosomal
of the plasmid inside the host bacterium may not hold in antibiotic resistance in bacteria: genetic transformation of
our case. Escherichia coli by R-factor. Proc. Natl. Acad. Sci. USA, 1972,
Another explanation regarding the difference in trans- 69, 2110–2114.
formation efficiency can be due to different levels of 2. Tsen, S. D., Fang, S. S., Chen, M. J., Chien, J. Y., Lee, C. C. and
concatemeric DNA formation during ligation. A con- Tsen, D. H., Natural plasmid transformation in Escherichia coli.
J. Biomed. Sci., 2002, 9, 246–252.
catemeric DNA is made up of intermolecular ligation be- 3. Baur, B., Hanselmann, K., Schlimme, W. and Jenni, B., Genetic
tween the same plasmid molecules which results in direct transformation in freshwater: Escherichia coli is able to develop
repeats. After entry into the cell, intramolecular recombi- natural competence. Appl. Environ. Microbiol., 1996, 62, 3673–
nation within the concatemer (that may be circular or 3678.
linear) results in the original plasmid, which can then 4. Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning:
A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold
multiply normally producing transformants. In our experi- Spring Harbor, NY, 1989.
ments, the three intense bands observed at the topmost 5. Sinden, R. R., DNA supercoiling. In DNA Structure and Function,
front of the gel comprise this category of DNA because (ed. Sinden, R. R.), Academic Press, 1994, pp. 95–133.
of their larger size. Does concatemeric DNA formation 6. Goldstein, E. and Drlica, K., Regulation of bacterial DNA super-
have any effect on the transformation efficiency we coiling: plasmid linking numbers vary with growth temperatures.
Proc. Natl. Acad. Sci. USA, 1984, 81, 4046–4050.
observed here? Entry of concatemeric DNA into compe- 7. Kumar, V., Transfromation in Escherichia coli: effect of heat
tent E. coli cells during heat shock transformation cannot shock of plasmid conformation. 2008. M Sc project report,
be ruled out. But in our case, even if the concatemeric Department of Molecular Biology and Biotechnology, Tezpur
DNA enters E. coli during transformation, possibility of University, Tezpur, 2008.
its influence on the transformation is unlikely because the 8. Bauer, F., Hertel, C. and Hammes, W. P., Transformation of
Escherichia coli in foodstuffs. Syst. Appl. Microbiol., 1999, 22,
competent cells used are recombination deficient (recA- 161–168.
deficient). Therefore, in this study, the explanation for 9. Singh, M., Yadav, A., Ma, X. and Amoah, E., Plasmid DNA trans-
varied levels of concatemeric DNA leading to different formation in Escherichia coli: effect of heat shock temperature,
transformation outcomes is irrelevant. duration, and cold incubation of CaCl2 treated cells. Int. J. Bio-
The findings obtained in this study with pBSKS, which technol. Biochem., 2010, 6, 561–568.
10. Pope, B. and Kent, H. M., High efficiency 5 min transformation of
is a small-sized plasmid of ~ 3.0 kb, might not hold for Escherichia coli. Nucleic Acids Res., 1996, 24, 536–537.
larger plasmids and this needs to be tested in future. The 11. Dorman, C. J. and Corcoran, C. P., Bacterial DNA topology and
differences in transformation efficiency of the ligated infectious disease. Nucleic Acids Res., 2009, 37, 672–678.
sample in various other temperatures also need to be 12. Dorman, C. J., DNA supercoiling and bacterial gene expression.
found. This is likely to give an insight into some of the Sci. Prog., 2006, 89, 151–166.
13. Conter, A., Plasmid DNA supercoiling and survival in long-term
interesting phenomena of DNA conformation during in cultures of Escherichia coli: role of NaCl. J. Bacteriol., 2003, 185,
vitro ligation and on the way through the cell envelope of 5324–5327.
E. coli during transformation and persistence of plasmids
with variable conformations inside the E. coli cell. It
might turn out in future that a relaxed state of a plasmid ACKNOWLEDGEMENTS. We thank the anonymous reviewer for
critical comments on the manuscript. We also thank Rocktatpal Kon-
is more efficient for transformation than that of a super- war, Tezpur University for his comments. P.K. and V.K. thank DBT,
coiled state, which will be completely different from our New Delhi for financial support and A.B. thanks the UGC, New Delhi
assumption made in Figure 1. Whether relaxed state of for providing Junior Research Fellowship. We also thank Dr R. V.
the plasmid is less efficient than the supercoiled state Sonti, CCMB, Hyderabad for the bacterial strain and plasmid used in
during transformation, is only a hypothesis proposed in this study.
this study considering the well-known fact that the Declaration. Bacterial strains used were non-pathogenic and were
relaxed plasmid migrates slower than the supercoiled sterilized after the experiment to avoid contamination in the environ-
plasmid during gel electrophoresis. Yet, none of the ment. Ethidium bromide use and disposal was performed following
available literature has cited experimental evidence statutory instructions.
regarding the transformation efficiency of relaxed and
Received 1 June 2012; revised accepted 8 January 2013
supercoiled plasmid. In fact, the obvious practical limita-
CURRENT SCIENCE, VOL. 104, NO. 6, 25 MARCH 2013 751