1. Letters in Applied Microbiology ISSN 0266-8254
ORIGINAL ARTICLE
A colony-to-lawn method for efficient transformation of
Escherichia coli
Y. An, A. Lv and W. Wu
Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, China
Keywords Abstract
chemical transformation, competent cells,
electroporation, low-copy-number plasmid, Aims: To develop a fast, convenient, inexpensive and efficient Escherichia coli
mutant library. transformation method for changing hosts of plasmids, which can also facilitate
the selection of positive clones after DNA ligation and transformation.
Correspondence Methods and Results: A single fresh colony from plasmid-containing donor
Wenfang Wu, Institute of Applied Ecology,
strain is picked up and suspended in 75% ethanol. Cells are pelleted and resus-
Chinese Academy of Sciences. No.72 Wenhua
Road. Shenyang 110016, China.
pended in CaCl2 solution and lysed by repetitive freeze–thaw cycles to obtain
E-mail: wshr100@sina.com.cn plasmid-containing cell lysate. The E. coli recipient cells are scraped from the
lawn of LB plate and directly suspended in the plasmid-containing cell lysate
2010 ⁄ 0411: received 11 March 2010, revised for transformation. Additionally, a process based on colony-to-lawn transfor-
4 April 2010 and accepted 21 April 2010 mation and protein expression was designed and conveniently used to screen
positive clones after DNA ligation and transformation.
doi:10.1111/j.1472-765X.2010.02864.x
Conclusions: With this method, a single colony from plasmid-containing
donor strain can be directly used to transform recipient cells scraped from
lawn of LB plate. Additionally, in combination with this method, screening of
positive clones after DNA ligation and transformation can be convenient and
time-saving.
Significance and Impact of the Study: Compared with current methods, this
procedure saves the steps of plasmid extraction and competent cell preparation.
Therefore, the method should be highly valuable especially for high-throughput
changing hosts of plasmids during mutant library creation.
efficiency (Okamoto et al. 1997; McCormac et al. 1998).
Introduction
In addition, a liposome-mediated transformation system
Changing hosts of plasmids by transformation is essential has been developed, because bacterial cells were found to
for many experiments in molecular biology, molecular be susceptible to transformation by liposomes (Kawata
genetics, etc. The CaCl2-mediated chemical transforma- et al. 2003). Although the methods described earlier have
tion is one of the most commonly used transformation provided various choices for efficient transformation of
methods until now. With this method, after treatment Escherichia coli, they are all dependent on the extraction
with CaCl2, a transient state of ‘competence’ is introduced of plasmid DNA beforehand. Therefore, when changing
to the recipient cells, and the cells are more likely to the hosts of hundreds or thousands of plasmids is
incorporate bacteriophage DNA or plasmid DNA (Man- performed, the work should be very time-consuming,
del and Higa 1970; Cohen et al. 1972; Oishi and Cosloy expensive and inconvenient.
1972). Some modified methods have been designed to In addition, during molecular cloning or construction
promote the efficiency of chemical transformation (Golub of mutant libaries, frameshift mutations often occur,
1988; Liu and Rashidbaigi 1990; Tang et al. 1994; Pope which may prevent the expression of proper proteins in
and Kent 1996; Chen et al. 2001; Zeng et al. 2006). E. coli. Although these mutations can be detected and
Another efficient transformation method is electro- removed by DNA sequencing of randomly selected clones,
poration, which can introduce a higher transformation the process is inconvenient especially when changing
ª 2010 The Authors
98 Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010) 98–103

2. Y. An et al. How to make transformation more efficient
hosts of multiple plasmids are performed during muta- BL21(DE3) harbouring pETM11-P450-BM3 obtained
tion library creation. To address these problems, we either from colony-to-lawn transformation or from chem-
describe a rapid, convenient and inexpensive method for ical transformation were cultured in TB media supple-
changing E. coli hosts of plasmids. Additionally, based on mented with kanamycin. The cultures were induced using
this method and protein expression, a process was IPTG (0Æ2 mmol l)1) at the exponential growth phase and
designed and conveniently used to screen positive clones incubated at 20°C with shaking at 150 rev min)1 over-
after DNA ligation and transformation. night. As a control, two colonies from the E. coli JM109
strain harbouring pETM11-P450-BM3 were also used for
induced protein expression as described earlier. Cells
Materials and methods
from these cultures were pelleted by centrifugation and
Escherichia coli JM109 strains harbouring plasmids pUC19 checked the expression levels of protein P450-BM3 by
(Ampr), pBR322 (Ampr), pYES2 (Ampr), pLysS (Camr), SDS-PAGE.
pSE380 (Ampr), pETM-11 (Kanr) and pETM11-p450BM3 A mutant library of P450-BM3 was generated by error-
(Kanr) were grown on antibiotic-supplemented LB agar prone PCR. The primers P450-For (5¢-GAGGGATACCA-
plates for 36 h. The concentrations of the antibiotics TGGCAATTAAAGAAATGCCTCAGCC-3¢) and P450-Rev
ampicillin, chloramphenicol and kanamycin were 50, 30 (5¢-CTCGCGGCCGCTTACCCAGCCCACACGTCTTTTG-
and 50 mg l)1 respectively. For each strain, a single CG-3¢) were used for PCR amplification. The PCR was
colony was carefully picked up without gouging the agar. performed in mixture containing 2 ng of P450-BM3 tem-
Each colony was suspended in a tube containing 200 ll plate DNA, 0Æ5 lmol l)1 both primers, 1 mmol l)1
Milli-Q water followed by the addition of 600 ll ethanol d(C ⁄ T)TP, 0Æ2 mmol l)1 d(A ⁄ G)TP, 40 nmol l)1 MgCl2,
to the tube. The mixtures were put in room temperature 1· Taq polymerase buffer and 3 Unit Taq polymerase
for 5 min, and then the cells were pelleted by centrifuga- with a total volume of 50 ll. This reaction mixture was
tion. The tubes were put upside down for 10 min at heated at 95°C for 2 min followed by 30 cycles of incuba-
room temperature to dry pellets, and a 30-ll aliquot of tion at 95°C for 1 min, 48°C for 40 s, and 72°C for
0Æ1 mol l)1 CaCl2 was added to each tube and mixed 5 min and a final incubation at 72°C for 10 min. After
carefully. Then, the cells of different strains were lysed by purification, the PCR product was digested with NotI and
frozen at )80°C and thawed at 100°C for three cycles to NcoI and cloned into the corresponding restriction
obtain plasmid-containing cell lysates. The recipient strain enzyme sites of pETM11 vector and transformed into
BL21(DE3) was intensively grown on LB agar plates for E. coli JM109. Ten randomly selected transformants were
24 h to form lawn. The cells from lawn were carefully used to transform E. coli recipient strain BL21(DE3) with
scraped without gouging the agar and resuspended in five the colony-to-lawn transformation method. After trans-
times volume of ice-cold water. A 30-ll aliquot of cells formation, transformed bacteria were grown in 50-ml
suspension was transferred to each tube containing the auto-inducing media (ZYM-5052) (Studier 2005). The
plasmid-containing cell lysate and mixed gently. The mix- cultures were first incubated at 37°C till OD600 = 1 and
tures were incubated on ice for 15 min followed by heat then incubated at 20°C overnight with shaking at
shock at 42°C for 40 s to perform transformation. Trans- 150 rev min)1. Cells from 5 ml of each culture were pel-
formed bacteria were grown and selected by standard leted by centrifugation and used to check protein expres-
methods. The number of transformants after each trans- sion by SDS-PAGE, and the remaining cultures (about
formation with a single colony of plasmid-containing 45 ml for each) were kept at 4°C. The plasmids were
donor strain was calculated after incubation at 37°C for extracted from the remaining cultures of positive clones
24 h. After each transformation, the plasmids were with expected protein expression, and DNA sequencing
extracted from five randomly selected transformants and was performed.
re-transformed into BL21(DE3) competent cells with the
traditional chemical transformation method. This was
Results
used to check whether the antibiotic-resistant colonies
were real transformants or just E. coli mutants or contam- The colony-to-lawn transformation method for changing
inants. As a control, the cell lysates were directly spread hosts of plasmids is illustrated in Fig. 1a. The first step is
on antibiotic-supplemented LB agar plates to check preparation of plasmid-containing cell lysate. A single
whether all the cells were sterilized after 75% ethanol colony from plasmid donor strain is suspended in 75%
incubation and freeze–thaw cycles. Changing hosts of ethanol followed by centrifugation to get pellet, and then
plasmid pETM11-P450-BM3 from JM109 to BL21(DE3) the cells are resuspended in CaCl2 solution and lysed
was also performed with chemical transformation after by freeze–thaw cycles to obtain plasmid-containing cell
plasmid extraction. Then, two transformants of lysate. The second step is preparation of recipient cells for
ª 2010 The Authors
Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010) 98–103 99

3. How to make transformation more efficient Y. An et al.
(a) (b) Colonies from Colonies from
Colonies from plasmid-containing
plasmid-containing plasmid-containing Lawn from
donor strain donor strain recipient strain
donor strain
Lawn from
recipient strain
A single colony Liquid
culture
200 µl water Cells pelleted by Cells scraped
600 µl ethanol centrifugation from lawn
Water Suspension Competent cell
preparation
30 µl CaCI2 Freeze-thaw
solution cycles Plasmid
extraction
Transformation Transformation
Transformation
Chemical transformation Colony-to-lawn transformation
(c) (d)
M 1 2 3 120
Transformation frequencies
100
120- 80
100-
85- 60
40
50-
20
(kDa) 0
pUC19 pBR322 pYES2 pLysS pSE380 pETM-11 pETM 11-
p450BM3
Plasmids
Figure 1 The colony-to-lawn transformation method used for changing hosts of plasmid. (a) Outline of the colony-to-lawn transformation
method. A single colony from plasmid donor strain is washed with 75% ethanol and air-dried, and then cells are suspended in CaCl2 solution and
lysed by freeze–thaw cycles to obtain plasmid-containing cell lysate. At the same time, cells of plasmid recipient strain are scraped carefully from
fresh lawn and suspended in ice-cold water. Then, the recipient cells and plasmid-containing cell lysate are mixed gently and performed transfor-
mation by heat shock method. The transformed bacteria are grown and selected by standard methods. (b) Comparison of the colony-to-lawn
transformation method and the chemical transformation method. Plasmid extraction and competent cell preparation are essential steps for chemi-
cal transformation, but not necessary for colony-to-lawn transformation. (c) SDS-PAGE gel shows protein expression of P450-BM3 before and
after changing hosts of pETM11-P450-BM3 either by colony-to-lawn transformation or by chemical transformation. Lane M: protein molecular
weight marker; lane 1, after host changing of pETM11-P450-BM3 with the chemical transformation method; lanes 2, after host changing of
pETM11-P450-BM3 with the colony-to-lawn transformation method; lanes 3, before host changing of pETM11-P450-BM3 (i.e. protein expressed
in Escherichia coli JM109). (d) The numbers of transformants obtained by changing hosts of various plasmids with the colony-to-lawn transforma-
tion method. Each value represents the mean of five independent experiments.
transformation. Cells of E. coli recipient strain are scraped plasmid extraction and competent cell preparation steps
carefully from fresh lawn without gouging the agar and are needed (Fig. 1b). Using pETM11-P450-BM3 as a sam-
then suspended in ice-cold water. The third step is trans- ple, we changed its hosts from E. coli JM109 to
formation. An aliquot of recipient cells and plasmid-con- BL21(DE3) either by colony-to-lawn transformation or by
taining cell lysate are mixed gently and we performed chemical transformation. After IPTG induction, the simi-
transformation by heat shock method. Then, transformed lar expression levels of P450-BM3 protein were obtained
bacteria are grown and selected by standard methods. (Fig. 1c), indicating that there is no fundamental differ-
The colony-to-lawn transformation method is more ence between these transformants. We tested the colony-
convenient and rapid than current methods, because no to-lawn transformation method by using it to change the
ª 2010 The Authors
100 Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010) 98–103

4. Y. An et al. How to make transformation more efficient
hosts of various plasmids, including the low-copy-number containing cell lysate, indicating that 75% ethanol incuba-
plasmid pLysS. As a result, no less than 60 transformants tion and freeze–thaw cycles were efficient for sterilization,
were available after each transformation with a single col- and no transformants obtained after transformation were
ony of plasmid-containing donor strain (Fig. 1d). Addi- mutants or contaminants.
tionally, the method is very convenient, because the LB A process based on colony-to-lawn transformation and
agar plates with colonies of donor strains and recipient protein expression was designed and conveniently used to
strain can be stocked at 4°C for at least 7 days without remove frameshift mutations during the construction of
affecting the transformation obviously (data not shown). mutant library (Fig. 2a). Recombinant plasmids are
As a control, plasmids from the randomly selected trans- constructed and transformed into E. coli cloning strain,
formants were successfully re-transformed into E. coli followed by changing the hosts of plasmids from cloning
BL21(DE3) by chemical transformation, indicating that strain to expression strain with the colony-to-lawn trans-
the antibiotic-resistant colonies after colony-based trans- formation method. Then, randomly selected transfor-
formation were real transformants but not E. coli mutants mants are cultured in auto-inducing media overnight. An
or contaminants. In addition, no colony was found on the aliquot of each culture is used to check protein expression
antibiotic-containing agar plates spread with the plasmid- by SDS-PAGE, and only the positive clones having
(a) Construction of (b) Construction of
recombinant plasmid recombinant plasmid
Transformation
Transformation
Colonies of
Colonies of
plasmid-containing plasmid-containing
donor strain donor strain
Colony-based Overnight cultures
transformation derived from
One day single colonies
1 2 3 4
Liquid culture of Less than
recipient cells two days
1 2 3 4 1 3 Extract plasmid from
each clone
2 4
Transformation into
1 2 3 4 recipient cells
1
3 Induced expression
Extract plasmids from 1 2 3 4
positive clones SDS-Page analysis One day
1 2 3 4
DNA Sequencing or
functional analysis SDS-Page
analysis
DNA Sequencing or
functional analysis
Figure 2 Protein expression in combination with the colony-to-lawn transformation method to screen in-frame clones from mutant library. (a)
Outline of the experimental strategy. Plasmids from mutant library construction were changed hosts from cloning strain to expression strain with
the colony-to-lawn transformation method. Then, the randomly selected transformants are checked for protein expression by SDS-PAGE. Plasmids
are extracted for positive clones, and DNA sequencing or next round of mutagenesis was performed (shown as dotted line). (b) The chemical
transformation method used for the same purpose. Plasmids are extracted from randomly selected clones after mutant library construction and
transformed into competent cells of expression strain for protein expression and SDS-PAGE analysis. The plasmids extracted from the clones which
have expected protein expression are used for DNA sequencing or next round of mutagenesis (shown as dotted line).
ª 2010 The Authors
Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010) 98–103 101

5. How to make transformation more efficient Y. An et al.
expected protein expression are used to extract plasmids incorrect protein expression in E. coli. Therefore, expres-
from their remaining cultures, and DNA sequencing or sion of proteins (especially for the well expressed
another round of mutagenesis was performed Although proteins) can be used to predict whether the genes are
the current transformation methods can be used for the in-frame, which can be further determined by DNA
same purpose, the process should be less convenient, sequencing. This strategy is reasonable because less
because more time and an additional experimental step plasmids need to be extracted for DNA sequencing.
(competent cell preparation) are needed (Fig. 2b). Addi- Therefore, a process based on colony-to-lawn transforma-
tionally, more plasmids should to be extracted, because tion and protein expression provides a convenient way to
the clones used for plasmid extraction are before protein screen in-frame clones from mutant libraries.
expression screening. In this work, the recombinant plas- In conclusion, as a simple and convenient DNA trans-
mids with random mutations of P450-BM3 gene intro- formation strategy, this method may find wide applica-
duced by error-prone PCR were used to test this method. tions in bioscience and biotechnology, especially when
The recombinant plasmids were changed hosts from clon- changing hosts of multiple plasmids is needed.
ing strain JM109 to expression strain BL21(DE3) with the
colony-to-lawn transformation method. Then, ten randomly
Acknowledgements
selected transformants were used to check protein expres-
sion levels, five of them were found to have expected The authors thank Sergi Castellano and Promdonkoy
protein expression. The plasmids were extracted and Patcharee for helpful discussions and review of this man-
DNA sequencing was performed, and as a result, all the uscript.
DNA sequences of positive clones were found to be in the
correct open reading frames.
References
Chen, X., Guo, P., Xie, Z. and Shen, P. (2001) A
Discussion
convenient and rapid method for genetic transformation
With this method, 75% ethanol is used for suspension of of E. coli with plasmids. Antonie Van Leeuwenhoek 80,
the colony, because it has the functions of sterilization, 297–300.
DNA sedimentation and pellet washing at the same time. Cohen, S.N., Chang, A.C.Y. and Hsu, L. (1972) Nonchromo-
Therefore, this treatment can avoid contamination of the somal antibiotic resistance in bacteria: genetic transforma-
plasmid donor strain after transformation and at the tion of Escherichia coli by R-factor DNA. Proc Natl Acad
same time reduce the loss of plasmid DNA during pellet Sci U S A 69, 2110–2114.
Golub, E.I. (1988) ‘One minute’ transformation of competent
washing. It is worth noting that E. coli cells from colony
E. coli by plasmid DNA. Nucleic Acids Res 16, 1641.
are difficult to suspend directly in 75% ethanol, so the
Kawata, Y., Yano, S. and Kojima, H. (2003) Escherichia coli
cells should be first suspended in water and then in 75%
can be transformed by a liposome-mediated lipofection
ethanol by adding proper volume of ethanol to the sus-
method. Biosci Biotechnol Biochem 67, 1179–1181.
pension. In addition, the recipient cells are conveniently
Liu, H.Y. and Rashidbaigi, A. (1990) Comparison of various
prepared, and repeated washing and centrifugation steps competent cell preparation methods for high efficiency
for preparing competent cells are not indispensable. This DNA transformation. BioTechniques 8, 21.
is because the cells are grown on plate but not in liquid Mandel, M. and Higa, A. (1970) Calcium-dependent bacterio-
culture, and there is no need to remove residual medium phage DNA infection. J Mol Biol 53, 159–162.
from cell pellet by washing. Although only a small McCormac, A.C., Elliott, M.C. and Chen, D.F. (1998) A simple
number of transformants can be obtained after colony-to- method for the production of highly competent cells of
lawn transformation, in fact the number of transformants Agrobacterium for transformation via electroporation. Mol
is not a limiting factor for changing hosts of plasmids in Biotechnol 9, 155–159.
most cases. It is because even thousands of transformants Oishi, M. and Cosloy, S.D. (1972) The genetic and biochemi-
can be obtained after transformation, and only one of cal basis of the transformability of Escherichia coli K12.
them is needed for the subsequent experiments. Because Biochem Biophys Res Commun 49, 1568–1572.
of its simplicity and convenience, the method should be Okamoto, A., Kosugi, A., Koizumi, Y., Yanagida, F. and
valuable especially for high-throughput changing hosts of Udaka, S. (1997) High efficiency transformation of Bacillus
plasmids during mutant library creation and functional brevis by electroporation. Biosci Biotechnol Biochem 61,
analysis. 202–203.
Frameshift mutations often occur during molecular Pope, B. and Kent, H.M. (1996) High efficiency 5 min trans-
cloning or construction of mutant libraries. It is worth formation of Escherichia coli. Nucleic Acids Res 24, 536–
noting that frameshift mutations can introduce no or 537.
ª 2010 The Authors
102 Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010) 98–103

6. Y. An et al. How to make transformation more efficient
Studier, F.W. (2005) Protein production by auto-induction transformation of E. coli. Nucleic Acids Res 22, 2857–
in high-density shaking cultures. Protein Expr Purif 41, 2858.
207–234. Zeng, W., Deng, Y., Yang, Z., Yuan, W., Huang, W., Zhu, C.,
Tang, X., Nakata, Y., Li, H.O., Zhang, M., Gao, H., Bai, Y., Li, Y. et al. (2006) high transformation efficiency of
Fujita, A., Sakatsume, O., Ohta, T. et al. (1994) The Escherichia coli with plasmids by adding amino modified
optimization of preparations of competent cells for silica-nanoparticles. Biotechnology 5, 341–343.
ª 2010 The Authors
Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010) 98–103 103