a colony to-lawn method for efficient transformation of escherichia coli

ORIGINAL ARTICLE

A colony-to-lawn method for efficient transformation ofEscherichia coliY. An, A. Lv and W. Wu

Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, China

Introduction

Changing hosts of plasmids by transformation is essential

for many experiments in molecular biology, molecular

genetics, etc. The CaCl2-mediated chemical transforma-

tion is one of the most commonly used transformation

methods until now. With this method, after treatment

with CaCl2, a transient state of ‘competence’ is introduced

to the recipient cells, and the cells are more likely to

incorporate bacteriophage DNA or plasmid DNA (Man-

del and Higa 1970; Cohen et al. 1972; Oishi and Cosloy

1972). Some modified methods have been designed to

promote the efficiency of chemical transformation (Golub

1988; Liu and Rashidbaigi 1990; Tang et al. 1994; Pope

and Kent 1996; Chen et al. 2001; Zeng et al. 2006).

Another efficient transformation method is electro-

poration, which can introduce a higher transformation

efficiency (Okamoto et al. 1997; McCormac et al. 1998).

In addition, a liposome-mediated transformation system

has been developed, because bacterial cells were found to

be susceptible to transformation by liposomes (Kawata

et al. 2003). Although the methods described earlier have

provided various choices for efficient transformation of

Escherichia coli, they are all dependent on the extraction

of plasmid DNA beforehand. Therefore, when changing

the hosts of hundreds or thousands of plasmids is

performed, the work should be very time-consuming,

expensive and inconvenient.

In addition, during molecular cloning or construction

of mutant libaries, frameshift mutations often occur,

which may prevent the expression of proper proteins in

E. coli. Although these mutations can be detected and

removed by DNA sequencing of randomly selected clones,

the process is inconvenient especially when changing

Keywords

chemical transformation, competent cells,

electroporation, low-copy-number plasmid,

mutant library.

Correspondence

Wenfang Wu, Institute of Applied Ecology,

Chinese Academy of Sciences. No.72 Wenhua

Road. Shenyang 110016, China.

E-mail: [email protected]

2010 ⁄ 0411: received 11 March 2010, revised

4 April 2010 and accepted 21 April 2010

doi:10.1111/j.1472-765X.2010.02864.x

Abstract

Aims: To develop a fast, convenient, inexpensive and efficient Escherichia coli

transformation method for changing hosts of plasmids, which can also facilitate

the selection of positive clones after DNA ligation and transformation.

Methods and Results: A single fresh colony from plasmid-containing donor

strain is picked up and suspended in 75% ethanol. Cells are pelleted and resus-

pended in CaCl2 solution and lysed by repetitive freeze–thaw cycles to obtain

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

for transformation. Additionally, a process based on colony-to-lawn transfor-

mation and protein expression was designed and conveniently used to screen

positive clones after DNA ligation and transformation.

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.

Letters in Applied Microbiology ISSN 0266-8254

98 Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010) 98–103

ª 2010 The Authors

hosts of multiple plasmids are performed during muta-

tion library creation. To address these problems, we

describe a rapid, convenient and inexpensive method for

changing E. coli hosts of plasmids. Additionally, based on

this method and protein expression, a process was

designed and conveniently used to screen positive clones

after DNA ligation and transformation.

Materials and methods

Escherichia coli JM109 strains harbouring plasmids pUC19

(Ampr), pBR322 (Ampr), pYES2 (Ampr), pLysS (Camr),

pSE380 (Ampr), pETM-11 (Kanr) and pETM11-p450BM3

(Kanr) were grown on antibiotic-supplemented LB agar

plates for 36 h. The concentrations of the antibiotics

ampicillin, chloramphenicol and kanamycin were 50, 30

and 50 mg l)1 respectively. For each strain, a single

colony was carefully picked up without gouging the agar.

Each colony was suspended in a tube containing 200 ll

Milli-Q water followed by the addition of 600 ll ethanol

to the tube. The mixtures were put in room temperature

for 5 min, and then the cells were pelleted by centrifuga-

tion. The tubes were put upside down for 10 min at

room temperature to dry pellets, and a 30-ll aliquot of

0Æ1 mol l)1 CaCl2 was added to each tube and mixed

carefully. Then, the cells of different strains were lysed by

frozen at )80�C and thawed at 100�C for three cycles to

obtain plasmid-containing cell lysates. The recipient strain

BL21(DE3) was intensively grown on LB agar plates for

24 h to form lawn. The cells from lawn were carefully

scraped without gouging the agar and resuspended in five

times volume of ice-cold water. A 30-ll aliquot of cells

suspension was transferred to each tube containing the

plasmid-containing cell lysate and mixed gently. The mix-

tures were incubated on ice for 15 min followed by heat

shock at 42�C for 40 s to perform transformation. Trans-

formed bacteria were grown and selected by standard

methods. The number of transformants after each trans-

formation with a single colony of plasmid-containing

donor strain was calculated after incubation at 37�C for

24 h. After each transformation, the plasmids were

extracted from five randomly selected transformants and

re-transformed into BL21(DE3) competent cells with the

traditional chemical transformation method. This was

used to check whether the antibiotic-resistant colonies

were real transformants or just E. coli mutants or contam-

inants. As a control, the cell lysates were directly spread

on antibiotic-supplemented LB agar plates to check

whether all the cells were sterilized after 75% ethanol

incubation and freeze–thaw cycles. Changing hosts of

plasmid pETM11-P450-BM3 from JM109 to BL21(DE3)

was also performed with chemical transformation after

plasmid extraction. Then, two transformants of

BL21(DE3) harbouring pETM11-P450-BM3 obtained

either from colony-to-lawn transformation or from chem-

ical transformation were cultured in TB media supple-

mented with kanamycin. The cultures were induced using

IPTG (0Æ2 mmol l)1) at the exponential growth phase and

incubated at 20�C with shaking at 150 rev min)1 over-

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

from these cultures were pelleted by centrifugation and

checked the expression levels of protein P450-BM3 by

SDS-PAGE.

A mutant library of P450-BM3 was generated by error-

prone PCR. The primers P450-For (5¢-GAGGGATACCA-

TGGCAATTAAAGAAATGCCTCAGCC-3¢) and P450-Rev

(5¢-CTCGCGGCCGCTTACCCAGCCCACACGTCTTTTG-

CG-3¢) were used for PCR amplification. The PCR was

performed in mixture containing 2 ng of P450-BM3 tem-

plate DNA, 0Æ5 lmol l)1 both primers, 1 mmol l)1

d(C ⁄ T)TP, 0Æ2 mmol l)1 d(A ⁄ G)TP, 40 nmol l)1 MgCl2,

1· Taq polymerase buffer and 3 Unit Taq polymerase

with a total volume of 50 ll. This reaction mixture was

heated at 95�C for 2 min followed by 30 cycles of incuba-

tion at 95�C for 1 min, 48�C for 40 s, and 72�C for

5 min and a final incubation at 72�C for 10 min. After

purification, the PCR product was digested with NotI and

NcoI and cloned into the corresponding restriction

enzyme sites of pETM11 vector and transformed into

E. coli JM109. Ten randomly selected transformants were

used to transform E. coli recipient strain BL21(DE3) with

the colony-to-lawn transformation method. After trans-

formation, transformed bacteria were grown in 50-ml

auto-inducing media (ZYM-5052) (Studier 2005). The

cultures were first incubated at 37�C till OD600 = 1 and

then incubated at 20�C overnight with shaking at

150 rev min)1. Cells from 5 ml of each culture were pel-

leted by centrifugation and used to check protein expres-

sion by SDS-PAGE, and the remaining cultures (about

45 ml for each) were kept at 4�C. The plasmids were

extracted from the remaining cultures of positive clones

with expected protein expression, and DNA sequencing

was performed.

Results

The colony-to-lawn transformation method for changing

hosts of plasmids is illustrated in Fig. 1a. The first step is

preparation of plasmid-containing cell lysate. A single

colony from plasmid donor strain is suspended in 75%

ethanol followed by centrifugation to get pellet, and then

the cells are resuspended in CaCl2 solution and lysed

by freeze–thaw cycles to obtain plasmid-containing cell

lysate. The second step is preparation of recipient cells for

Y. An et al. How to make transformation more efficient

ª 2010 The Authors

Journal compilation ª 2010 The Society for Applied Microbiology, Letters in Applied Microbiology 51 (2010) 98–103 99

transformation. Cells of E. coli recipient strain are scraped

carefully from fresh lawn without gouging the agar and

then suspended in ice-cold water. The third step is trans-

formation. An aliquot of recipient cells and plasmid-con-

taining cell lysate are mixed gently and we performed

transformation by heat shock method. Then, transformed

bacteria are grown and selected by standard methods.

The colony-to-lawn transformation method is more

convenient and rapid than current methods, because no

plasmid extraction and competent cell preparation steps

are needed (Fig. 1b). Using pETM11-P450-BM3 as a sam-

ple, we changed its hosts from E. coli JM109 to

BL21(DE3) either by colony-to-lawn transformation or by

chemical transformation. After IPTG induction, the simi-

lar expression levels of P450-BM3 protein were obtained

(Fig. 1c), indicating that there is no fundamental differ-

ence between these transformants. We tested the colony-

to-lawn transformation method by using it to change the

Colonies from Colonies from Colonies fromplasmid-containing plasmid-containing plasmid-containing

recipient strain

recipient strain

Cells pelleted bycentrifugation

Cells scraped

Suspension

Freeze-thawcyclessolution

A single colony

100

80

pUC19 pBR322 pLysS pSE380 pETM-11 pETM 11-p450BM3

pYES2

20

0

40

60

120

100-85-

50-

(kDa)

120-

Colony-to-lawn transformationChemical transformation

Transformation

Plasmids

Tra

nsfo

rmat

ion

freq

uenc

ies

Transformation

Competent cellpreparation

Liquidculture

extractionPlasmid

Transformation

M 1 2 3

Water

600 µl ethanol

30 µl CaCI2

200 µl water

Lawn from

from lawn

Lawn from

donor straindonor strain donor strain

(a) (b)

(c) (d)

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.

How to make transformation more efficient Y. An et al.


ª 2010 The Authors

hosts of various plasmids, including the low-copy-number

plasmid pLysS. As a result, no less than 60 transformants

were available after each transformation with a single col-

ony of plasmid-containing donor strain (Fig. 1d). Addi-

tionally, the method is very convenient, because the LB

agar plates with colonies of donor strains and recipient

strain can be stocked at 4�C for at least 7 days without

affecting the transformation obviously (data not shown).

As a control, plasmids from the randomly selected trans-

formants were successfully re-transformed into E. coli

BL21(DE3) by chemical transformation, indicating that

the antibiotic-resistant colonies after colony-based trans-

formation were real transformants but not E. coli mutants

or contaminants. In addition, no colony was found on the

antibiotic-containing agar plates spread with the plasmid-

containing cell lysate, indicating that 75% ethanol incuba-

tion and freeze–thaw cycles were efficient for sterilization,

and no transformants obtained after transformation were

mutants or contaminants.

A process based on colony-to-lawn transformation and

protein expression was designed and conveniently used to

remove frameshift mutations during the construction of

mutant library (Fig. 2a). Recombinant plasmids are

constructed and transformed into E. coli cloning strain,

followed by changing the hosts of plasmids from cloning

strain to expression strain with the colony-to-lawn trans-

formation method. Then, randomly selected transfor-

mants are cultured in auto-inducing media overnight. An

aliquot of each culture is used to check protein expression

by SDS-PAGE, and only the positive clones having

Construction of Construction ofrecombinant plasmid recombinant plasmid

Transformation

Transformation into

Transformation

Liquid culture of Less thanrecipient cells

recipient cells

Induced expression

One day

One day

two days

2

2

3

3

3

4

4

SDS-Page analysis

SDS-Page analysis

DNA Sequencing or

DNA Sequencing or

functional analysis

functional analysis

Extract plasmids from positive clones

1

2 3 41

1

1

2 3 41

2

2

3

3

4

4

1

1

donor strain donor strain

each cloneExtract plasmid from

single coloniesderived from

Overnight cultures

plasmid-containing plasmid-containing

Colonies of Colonies of

transformationColony-based

(a) (b)

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


expected protein expression are used to extract plasmids

from their remaining cultures, and DNA sequencing or

another round of mutagenesis was performed Although

the current transformation methods can be used for the

same purpose, the process should be less convenient,

because more time and an additional experimental step

(competent cell preparation) are needed (Fig. 2b). Addi-

tionally, more plasmids should to be extracted, because

the clones used for plasmid extraction are before protein

expression screening. In this work, the recombinant plas-

mids with random mutations of P450-BM3 gene intro-

duced by error-prone PCR were used to test this method.

The recombinant plasmids were changed hosts from clon-

ing strain JM109 to expression strain BL21(DE3) with the

colony-to-lawn transformation method. Then, ten randomly

selected transformants were used to check protein expres-

sion levels, five of them were found to have expected

protein expression. The plasmids were extracted and

DNA sequencing was performed, and as a result, all the

DNA sequences of positive clones were found to be in the

correct open reading frames.

Discussion

With this method, 75% ethanol is used for suspension of

the colony, because it has the functions of sterilization,

DNA sedimentation and pellet washing at the same time.

Therefore, this treatment can avoid contamination of the

plasmid donor strain after transformation and at the

same time reduce the loss of plasmid DNA during pellet

washing. It is worth noting that E. coli cells from colony

are difficult to suspend directly in 75% ethanol, so the

cells should be first suspended in water and then in 75%

ethanol by adding proper volume of ethanol to the sus-

pension. In addition, the recipient cells are conveniently

prepared, and repeated washing and centrifugation steps

for preparing competent cells are not indispensable. This

is because the cells are grown on plate but not in liquid

culture, and there is no need to remove residual medium

from cell pellet by washing. Although only a small

number of transformants can be obtained after colony-to-

lawn transformation, in fact the number of transformants

is not a limiting factor for changing hosts of plasmids in

most cases. It is because even thousands of transformants

can be obtained after transformation, and only one of

them is needed for the subsequent experiments. Because

of its simplicity and convenience, the method should be

valuable especially for high-throughput changing hosts of

plasmids during mutant library creation and functional

analysis.

Frameshift mutations often occur during molecular

cloning or construction of mutant libraries. It is worth

noting that frameshift mutations can introduce no or

incorrect protein expression in E. coli. Therefore, expres-

sion of proteins (especially for the well expressed

proteins) can be used to predict whether the genes are

in-frame, which can be further determined by DNA

sequencing. This strategy is reasonable because less

plasmids need to be extracted for DNA sequencing.

Therefore, a process based on colony-to-lawn transforma-

tion and protein expression provides a convenient way to

screen in-frame clones from mutant libraries.

In conclusion, as a simple and convenient DNA trans-

formation strategy, this method may find wide applica-

tions in bioscience and biotechnology, especially when

changing hosts of multiple plasmids is needed.

Acknowledgements

The authors thank Sergi Castellano and Promdonkoy

Patcharee for helpful discussions and review of this man-

uscript.

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a colony to-lawn method for efficient transformation of escherichia coli

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