Competent Cell Compendium:
Tools and Tips for Successful Transformations
• Competent Cell
Genoytpes and What
They Mean
• Selecting the
Right Cloning or
Expression Strain
• Calculating
Transformation
Efficiencies
• Cloning and
Expression
Protocols
iii
Table of Contents
Section 1: What are Competent Cells? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Definition of Competent Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Calculating Transformation Efficiencies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Section 2: Strains of Competent Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Cloning Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Selecting the Right Cloning Strain for Your Needs . .
Cloning Strains Available from Sigma-Aldrich . . . . . .
Expression Strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Selecting the Right Expression Strain for Your Needs
Expression Strains Available from Sigma-Aldrich . . . .
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3
3
3
6
6
7
Section 3: Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
What is Important in a Transformation? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
Forms of DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Amount of DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Source of DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Impurities in DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Storage and Handling of Competent Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Section 4: Transformation Protocols – Cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Chemical Transformation .
What Is Important
Protocol . . . . . . .
Electroporation . . . . . . . .
What Is Important
Protocol . . . . . . .
FAQs – Cloning . . . . . . . .
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11
11
12
13
13
14
15
Section 5: Transformation Protocols – Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Making a Stock Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
General Protein Expression Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
FAQs – Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Appendix 1: Genotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Appendix 2: Supporting Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Acknowledgements and General References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
www.sigma-aldrich.com
We would like to thank GeneChoice, Inc. for their support and content in the Competent Cell Compendium: Tools and Tips for Successful Transformation.
1
E
fficient uptake of nucleic acid by transformation of competent E. coli is an integral step in
molecular cloning and is required in order to perform numerous downstream applications such
as recombinant protein expression and mutagenesis. In order to transform, competent cells are
required. There are a number of commercially available competent cell strains and choosing the
optimal stain can be difficult. The Sigma-Aldrich Competent Cell Compendium: Tools and Tips for
Successful Transformation is intended to aid in the selection of the best strain for your needs.
WHAT ARE COMPETENT CELLS?
Competent cells are E. coli cells that have been specially
treated to transform efficiently. There are two types
of competent cells: chemically competent and
electrocompetent. If plasmid is simply added to
E. coli, nothing happens! The cells must be competent!
Add plasmid to E. coli
E. coli host cell
Plasmid
Result: NOTHING! The cells are not competent.
Chemically Competent
Chemically competent cells are treated with a buffer that
contains CaCl2 and other salts that disrupt the cell
membrane creating “holes” that allow the plasmids to pass
into the cell. Most researchers use chemically competent
cells because they are less expensive, can be made in the
lab and do not require special equipment.
Add plasmid to chemically competent E. coli
Chemically
competent
E. coli host cell
Plasmid
Result: E. coli transformed cell
Add plasmid to electrocompetent E. coli
Electrocompetent
E. coli host cell
Plasmid
Place in strong
electric field
-
+
Result: E. coli transformed cell
www.sigma-aldrich.com
Electrocompetent
Electrocompetent transformations require a high density of
cells and a non-ionic buffer. The cells are placed in an
electroporation device that delivers a pulse of electricity to
disrupt the membranes of the cells allowing the plasmids
to enter the cells. Electrocompetent formats provide the
highest transformation efficiencies, but do require an
electroporation device.
2
Section 1: WHAT ARE COMPETENT CELLS?
How Do I Calculate Transformation Efficiency?
Transformation efficiencies are based on the fraction of
cells that can be transformed. No matter how much DNA
is added, only a certain number of cells can be transformed.
This fraction of the cells is called the “fraction competent.”
The total number of transformants generated from a
reaction is determined by the number of cells present
and the fraction competent.
The following are typical transformation efficiencies:
106 = value efficiency cloning strains and BL21
expression strains
107 = general cloning purposes
Calculating Transformation Efficiency
Transformation efficiency is measured as the number
of transformants per microgram of pUC19 DNA.
Transformation efficiency is determined by dividing
the number of transformants (A) by the amount of
plasmid DNA (B).
1010
109
108
Total Transformants
Competent cells have a range of transformation
efficiencies. Transformation efficiency is a measure of how
well the cells incorporate and duplicate DNA of interest.
Transformation efficiency is measured in cfus, or Colony
Forming Units, per input DNA. The unofficial standard is
cfu/µg of pUC19 DNA.
A
107
106
105
104
Thunderbolt™
High Efficiency
Value Efficiency
B
103
102
0.001
109 = high efficiency grade
0.01
0.1
1
10
100
1,000 10,000 100,000
ng pUC19 DNA per reaction
1010 = highest efficiency (electrocompetent)
www.sigma-aldrich.com
Value efficiency cells are suitable for most purposes. For
instance, when transforming purified plasmid DNA or
expressing after cloning has already been done in a
cloning strain, value efficiency cells are sufficient. High
efficiency cells are excellent for everything from general
cloning to difficult cloning and libraries.
Electrocompetent cells are suitable for most purposes
with an efficiency of 1010.
Example:
A/B = 107/1 ng = 107/0.001 µg = 1010
Note: Transformation efficiency is determined in the linear part of the curve.
These are the fraction competent cells. In the non-linear portion of the curve
are cells that cannot be transformed.
3
Section 2: STRAINS OF COMPETENT CELLS
There are many strains of competent cells that are grouped into cloning strains and expression strains.
Each strain has beneficial properties. Selecting the strain most suitable for your application will provide
optimal results.
Cloning Strains
Competent cells most often used in cloning and
sequencing, but that are not well suited to gene
expression work, are referred to as cloning strains. There
are many different strains of E. coli available for cloning,
nearly all of them derived from a single strain called K12
which was first isolated in 1922. Since 1922, many
mutants have been made, resulting in the average strain
containing a long list of genetic markers (the genotype).
The Ideal Cloning Host
Ideal traits of cloning strains are listed below. For a detailed
explanation of these traits and others, please see page 20
Appendix 1: Genotypes.
hsdRk–
mcr-mrr
recA
endA
lacZ∆M15
does not restrict unmethylated DNA
does not restrict methylated DNA
does not recombine homologous DNA
does not degrade DNA
blue/white screening
tonA, T1R
resistant to T1 phage and its relatives
Cloning Strains from Sigma-Aldrich
Cloning Strain Selection Guide
Features
Strain background
GC5™
GC10™
Thunderbolt™ GC10™
JM109
HB101
K12
K12
K12
K12
K12xB
9
9
10
8
Transformation efficiency
10
10
10
10
108
Blue/White selection
Yes
Yes
Yes
Yes
No
Recombination deficient (recA)
Yes
Yes
Yes
Yes
Yes
Endonuclease deficient (endA)
Yes
Yes
Yes
Yes
No
Restriction deficient (hsdRK-)
Yes
Yes
Yes
Yes
Yes
Methyl restriction deficient (mcr-mrr )
No
Yes
Yes
Yes
No
Phage resistant (tonA, T1 )
Yes
Yes
Yes
No
No
F′ episome for single strand rescue
No
No
No
Yes
No
Q
lacI (higher level of lac repressor)
No
No
No
Yes
No
Ideal for:
cloning larger
plasmids and
genomic DNA
cloning
methylated and
genomic DNA
cloning
methylated and
genomic DNA
generation of
high-quality
plasmid and
single-stranded
DNA
popular strain,
cloning with
pBR322 & other
vectors without
α-complementation
R
www.sigma-aldrich.com
Sigma-Aldrich offers a variety of cloning strains. The table below features available cloning strains, their efficiencies, their traits,
and the ideal uses for each strain, to aid in the selection of the optimal strain.
4
Section 2: STRAINS OF COMPETENT CELLS
Cloning Strains from Sigma-Aldrich
GC5™ Chemically Competent Cells
Comparable to: DH5α™
Uses: plasmid propagation, cDNA library generation from
plasmid based vectors, and general cloning protocols.
Ideal for: larger plasmids.
Transformation Efficiency: >1 X 109 cfu/µg when transformed with non-saturating amounts of pUC19 control DNA.
Beneficial
recA
endA
lacZ∆M15
T1R
Traits:
increases plasmid stability
improves the quality of plasmid preparations
blue/white screening
phage resistance
GC10™ Chemically Competent Cells
Comparable to: DH10B™
Uses: cDNA library generation from plasmid-based vectors,
construction of gene banks and general cloning protocols.
Ideal for: cloning methylated and genomic DNA. Elimination
of host restriction systems allows construction of more
representative genomic libraries (allows cloning of
methlylated DNA).
Transformation efficiency: >1 X 109 cfu/µg when transformed with non-saturating amounts of pUC19 control DNA.
Genotype of GC5: F –Φ80lacZ∆M15∆(lacZYA-argF)U169
endA1 recA1 relA1 gyrA96 hsdR17 (rk–,mk+) phoA supE44
thi-1 λ – T1R
Package Sizes: GC5 competent cells are available in
convenient unipacks for one time use, standard aliquots
and in a 96-well plate format.
Cat. No.
G3169
G3044
G7419
Comparable to: DH10B™
Uses: cDNA library generation from plasmid-based vectors,
construction of gene banks and general cloning protocols.
Size
10 x 50 µl
20 x 50 µl
5 x 200 µl
1 EA
4 EA
Genotype of GC10: F –mcrA∆(mrr-hsdRMS-mcrBC)
Φ80dlacZ∆M15 ∆lacX74 endA1 recA1 ∆(ara, leu)7697
araD139 galU galK nupG rpsL λ–T1R
Package Sizes: GC10 chemically competent cells are available
in convenient unipacks for one time use and in standard
aliquots.
Cat. No.
G2919
G2794
Beneficial Traits:
recA
increases plasmid stability
endA
improves the quality of plasmid preparations
lacZ∆M15 blue/white screening
mcrA, mrr does not restrict methylated DNA
T1R
phage resistance
Thunderbolt™ GC10™ Electrocompetent Cells
Description
GC5 Chemically Competent
Cells, Unipack
GC5 Chemically Competent
Cells, Standard Aliquots
GC5 Chemically Competent
Cells, 96-Well Plates
Description
GC10 Chemically Competent
Cells, Unipack
GC10 Chemically Competent
Cells, Standard Aliquots
Size
10 x 50 µl
20 x 50 µl
5 x 200 µl
Genotype of GC10: F –mcrA∆(mrr-hsdRMS-mcrBC)
Φ80dlacZ∆M15 ∆lacX74 endA1 recA1 ∆(ara, leu)7697
ara∆139 galU galK nupG rpsL λ-T1R
Package Sizes: Thunderbolt GC10 electrocompetent cells
are only available in standard aliquots.
www.sigma-aldrich.com
Ideal for: cloning methylated and genomic DNA.
Transformation Efficiency: >1 X 1010 cfu/µg when transformed with non-saturating amounts of pUC19 control DNA.
Cat. No.
T7699
Description
Thunderbolt GC10 Electrocompetent Cells, Standard Aliquots
Beneficial Traits:
recA
increases plasmid stability
endA
improves the quality of plasmid preparations
lacZ∆M15 blue/white screening
mcrA, mrr does not restrict methylated DNA
T1R
phage resistance
GC5™, GC10™, and Thunderbolt™ are trademarks of GeneChoice®, Inc. DH5α™ and DH10B™ are trademarks of Invitrogen.
Size
5 x 80 µl
5 x 100 µl
5
Section 2: STRAINS OF COMPETENT CELLS
Cloning Strains from Sigma-Aldrich
JM109 Competent Cells
Uses: generation of high-quality plasmid and
single-stranded DNA.
Ideal for: generation of high quality plasmid and
single-stranded DNA.
Transformation Efficiency: >1 X 108 cfu/µg when
transformed with non-saturating amounts of pUC19
control DNA.
Genotype of JM109: F′ (traD36, proAB+ lacIq, lacZ∆M15)
endA1 recA1 hsdR17(rk–,mk+) mcrA supE44 λ- gyrA96 relA1
∆(lac-proAB) thi-1 lon
Package Size: JM109 competent cells are available in the
convenient unipack format for single use.
Cat. No.
J3895
Description
JM109 Competent Cells, Unipack
Size
10 x 50 µl
Beneficial Traits:
recA
increases plasmid stability
endA
improves quality of plasmid preparations
hsdR17
prevents the cleavage of heterologous DNA by
an endogenous endonuclease
F′ episome single strand stability
lacZ∆M15 blue/white screening
T1R
phage resistance
HB101 Competent Cells
Uses: routine subcloning and construction of cDNA libraries.
Ideal for: classic strain for general cloning purposes and also
for cloning genomic DNA.
Transformation Efficiencies: >1 x 108 cfu/µg when
transformed with non-saturating amounts of pUC19 control
DNA.
Genotype of HB101: F-, hsdS20(rB–,mB–), xyl5, λ–, recA13,
galK2, ara14, supE44, lacY1, rpsL20(str r), leuB6, mtl-1
Package Size: HB101 competent cells are provided in the
convenient unipack format for single use transformations.
Cat. No.
H3788
Description
HB101 Competent Cells, Unipack
Size
10 x 50 µl
www.sigma-aldrich.com
Beneficial Traits:
HB101 is a hybrid K12 x B strain.
recA13
minimizes recombination and aids in
insert stability
hsdS20(rB–,mB–) prevents cleavage of cloned DNA by
endogenous restriction
6
Section 2: STRAINS OF COMPETENT CELLS
Expression Strains
Expression strains are used to express a protein efficiently
from a given construct. There are two points to consider
when choosing an expression strain: the type of promoter
system being used and the level of promoter control
required.
T7 Promoter System
The first consideration is the type of promoter system
being used. One of the most common systems is the T7
promoter system. The T7 promoter system works by
utilizing T7 RNA polymerase to drive expression. Upon
induction with IPTG, T7 RNA polymerase is made by the
DE3 element in the chromosome. T7 RNA polymerase then
recognizes the T7 promoter on the clone and leads to the
production of RNA. Next, the RNA is translated into the
protein. If a T7 promoter system is being used, BL21(DE3)
strains are recommended. The DE3 designation indicates
that the strain is lysogenic for a lambda prophage
containing the inducible T7 RNA polymerase.
www.sigma-aldrich.com
If the system does not require T7 RNA polymerase to drive
expression, then the BL21 strain is a suitable choice. BL21
is an all-purpose expression strain directed by various
expression vector systems such as, lac, trc, tac, λPL and
araD. The strain naturally lacks two key proteases, lon and
ompT. The absence of proteolytic activity from lon and
ompT may reduce the degradation of some heterologous
proteins expressed in the strain.
Cloning in BL21
BL21 competent cells are traditionally referred to as
“expression strains.” Cloning can be performed in BL21, but
success depends on how efficient the cloning is. If using a
cloning method with high yield and low background, then
transformation can be done directly into BL21 to save time.
If cutting, pasting, and screening, it is recommended to use
the GC5 cloning strain to generate the construct and then
put the constructed plasmid into BL21.
BL21 Geneotypes
The genotypes of BL21 and phage λDE3 are listed below.
Note that the genotype of BL21(DE3) is just BL21 with the
phage DE3 added.
BL21
F-, ompT, hsdS(r–B,m–B), gal, dcm, Ion
λDE3
lacI, lacUV5-T7 gene 1, ind1, sam7, nin5
BL21(DE3)
F-, ompT, hsdS(r–B,m–B), gal, dcm, λDE3 (lacI,
lacUV5-T7 gene 1, ind1, sam7, nin5)
The Ideal Expression Strain
The ideal expression strain has the following traits:
endA
does not degrade DNA
hsd
does not restrict unmethylated DNA
lacIq
better control over the lac promoter
lon
lacks intracellular protease
Promoter Control
ompT
lacks extracellular protease
Additional BL21(DE3) strains are available depending on
the level of promoter control required. For instance, in the
T7 system, T7 RNA polymerase expression is repressed by
lacl, but a small amount of T7 RNA polymerase is still
produced even without IPTG induction and with the
presence of the lacIq allele. If the expressed protein is toxic,
even the low level of protein expression is enough to make
the cells sick. As a result, when producing a toxic protein,
increased promoter control is vital. BL21(DE3) pLysS or
pLysE competent cells are recommended for increased
promoter control in T7 systems. BL21(DE3)pLysS
competent cells express T7 lysozyme which is a natural
inhibitor of T7 RNA polymerase. This allows for improved
transcriptional control and reduction of “leaky” expression.
BL21(DE3)pLysE competent cells also express the T7
lysozyme, but at higher levels than BL21(DE3)pLysS. This
allows for a greater level of control over transciption and a
greater reduction in “leaky” expression. This strain is
usually required if the protein to be expressed is toxic to
the cell.
pLysS
inhibits T7 RNA polymerase
tonA
resistant to T1 phage
trfA
replicated oriV plasmids
T7 RNA Polymerase
To learn more about the traits listed here and about
other traits of expression strains, please see page 20
Appendix 1: Genotypes.
7
Section 2: STRAINS OF COMPETENT CELLS
Expression Strains from Sigma-Aldrich
Sigma-Aldrich offers BL21 strains with or without T7 RNA Polymerase and with varying levels of control over the lac promoter
(pLysS and pLysE). The guide below will aid in the selection of the ideal BL21 strain for your expression needs.
Features
BL21
BL21(DE3)
BL21(DE3) pLysS
BL21(DE3) pLysE
B
B
B
B
Strain background
Transformation efficiency
10
10
10
106
Restriction deficient (hsdSB)
Yes
Yes
Yes
Yes
6
lon and/or ompT protease deficient
7
6
ompT, lon
ompT, lon
ompT, lon
ompT, lon
T7 Polymerase
No
Yes
Yes
Yes
Deficient in cytosine methylation (dcm)
Yes
Yes
Yes
Yes
Deficient in galactose metabolism (gal)
Yes
Yes
Yes
Yes
T1 phage resistant
Yes
Yes
Yes
Yes
BL21-T1R
BL21(DE3)-T1R
Ideal for: high level production of heterologous proteins
directed by various expression vector systems (promoters
such as lac, trc, tac, λPL and araD).
Ideal for: high level induction and expression of genes
from any T7 promoter-based expression vector.
Transformation Efficiency: >1 x 10 cfu/µg when
transformed with non-saturating amounts of pUC19
control DNA.
6
Transformation Efficiency: >1 x 107 cfu/µg when
transformed with non-saturating amounts of pUC19
control DNA.
Beneficial Traits:
lon
reduces degradation of some heterologous
proteins expressed in the strain
DE(3) indicates that the strain is lysogenic for a lambda
prophage containing an inducible T7 RNA
polymerase under control of the lacUV5 promoter
ompT reduces degradation of some heterologous
proteins expressed in the strain
lon
tonA
ompT reduces degradation of some heterologous
proteins expressed in the strain
confers resistance to the lytic bacteriophages T1
and T5
reduces degradation of some heterologous
proteins expressed in the strain
Genotype of BL21-T1R: F –ompT hsdSB(rB–,mB–) gal dcm
tonA Ion
tonA
Package Size: BL21 competent cells are available in the
convenient unipack format for one time use.
Genotype of BL21(DE3)-T1R: F –ompT hsdSB(rB–mB–) gal
dcm λ(DE3) tonA Ion
Cat. No.
B2685
Description
BL21-T1R Competent
Cells, Unipack
Size
10 x 50 µl
confers resistance to the lytic bacteriophages T1
and T5
Package Size: BL21(DE3)-T1R competent cells are available
in the convenient unipack format for one time use.
Cat. No.
B2935
Description
BL21(DE3)-T1R Competent
Cells, Unipack
Size
10 x 50 µl
www.sigma-aldrich.com
Beneficial Traits:
8
Section 2: STRAINS OF COMPETENT CELLS
Expression Strains from Sigma-Aldrich
BL21(DE3)pLysS-T1R
BL21(DE3)pLysE-T1R
Ideal for: high level induction and expression of genes
from any T7 promoter-based expression vector.
Ideal for: high level induction and expression of genes
from any T7 promoter-based expression vector.
Transformation Efficiency: >5 x 106 cfu/µg when
transformed with non-saturating amounts of pUC19
control DNA.
Transformation Efficiency: >1 x 106 cfu/µg when
transformed with non-saturating amounts of pUC19
control DNA.
Beneficial Traits:
Beneficial Traits:
DE(3) indicates that the strain is lysogenic for a lambda
prophage containing an inducible T7 RNA
polymerase under control of the lacUV5 promoter
DE(3) indicates that the strain is lysogenic for a lambda
prophage containing an inducible T7 RNA
polymerase under control of the lacUV5 promoter
lon
lon
reduces degradation of some heterologous
proteins expressed in the strain
reduces degradation of some heterologous
proteins expressed in the strain
ompT reduces degradation of some heterologous
proteins expressed in the strain
ompT reduces degradation of some heterologous
proteins expressed in the strain
tonA
tonA
confers resistance to the lytic bacteriophages T1
and T5
pLysS The pLysS plasmid expresses T7 lysozyme, a natural
inhibitor of T7 polymerase allowing for improved
transcriptional control and reduction of “leaky”
expression. The pLysS plasmid also renders the cell
resistant to chloramphenicol (CmR) and contains
the p15A origin. This allows pLysS to be compatible
with plasmids containing the ColE1 or pMB1 origin.
Genotype of BL21(DE3)pLysS-T1R: F –ompT hsdSB(rB–,mB–)
gal dcm λ(DE3) tonA pLysS (CmR) Ion
Package Size: BL21(DE3)pLysS-T1R competent cells are
available in the convenient unipack format for one time use.
Cat. No.
B3310
Description
BL21(DE3)pLysS-T1R Competent
Cells, Unipack
Size
10 x 50 µl
confers resistance to the lytic bacteriophages T1 and T5
pLysE The pLysS plasmid expresses T7 lysozyme, a natural
inhibitor of T7 polymerase allowing for improved
transcriptional control and reduction of “leaky”
expression. The pLysE plasmid expresses T7 lysozme
at higher levels than the pLysS plasmid conferring a
greater level of control over the T7 polymerase.
This is usually only required when the recombinant
protein to be expressed may be toxic to the cell.
The pLysE plasmid also renders the cell resistant to
chloramphenicol (CmR) and contains the p15A
origin. This allows pLysE to be compatible with
plasmids containing the ColE1 or pMB1 origin.
Genotype of BL21(DE3)pLysE-T1R: F –ompT hsdSB(rB–,mB–)
gal dcm λ(DE3) tonA pLysE (CmR) Ion
Package Size: BL21(DE3)pLysE-T1R competent cells are
available in the convenient unipack format for one time use.
www.sigma-aldrich.com
Cat. No.
B3435
Description
BL21(DE3)pLysE-T1R Competent
Cells, Unipack
Size
10 x 50 µl
Visit sigma-aldrich.com/competentcells to view the presentation,
“Everthing You Need to Know About Competent Cells,” by Michael Smith, Ph.D.
9
Section 3: TRANSFORMATION
What Is Important in a Transformation?
After selecting the ideal competent cell strain, transformation is the next step. There are two ways to accomplish
transformation successfully: by chemical transformation or by electroporation. Regardless of whether the
transformation is being done for cloning or expression, the transformation procedures are the same.
Before beginning a transformation, it is important to know
what is required to achieve the best results. There are
many factors that affect transformation including the
following:
1. the form of DNA
2. the amount of DNA
3. the source of DNA
4. impurities in the DNA
per reaction, the non-transforming DNA will decrease the
efficiency of the transforming DNA approximately two-fold
for chemically competent cells and will not affect the
efficiency of electrocompetent cells.
If the ligation reaction is concentrated by precipitation and
500 ng of the ligation is added to a single reaction, the
competition effects can drop the transformation efficiency
ten-fold for chemically competent cells, but will not affect
electroporation.
5. storage and handling of the competent cells
Forms of DNA
Relaxed plasmids transform E. coli with the same efficiency
as supercoiled plasmids. Linear plasmids and single-stranded
plasmids transform very poorly (<1% as efficiently as double
strand circles). A special host is needed to achieve
chromosomal transformation, which is very inefficient.
Usually, a transformation involves a mixture of linear (nontransforming) and circular (transforming) DNA.
Source of DNA
DNA from eukaryotes is heavily methylated and E. coli has
restriction systems that restrict these types of methylation. As
a result, when cloning genomic DNA, it is recommended to
use a mcr mutant like GC10. DNA generated by PCR is
unmethylated, so cloning a PCR fragment from genomic
DNA does not require a mcr mutant.
Impurities in DNA
Adding more DNA to a transformation does not necessarily
lead to more transformants. For chemically competent cells,
adding more than 10 ng of pUC19 DNA does not result in
significantly more transformants. The point of diminishing
returns is about 100 ng of pUC19 for electrocompetent cells.
With ligations, a ligation will have insert DNA, linear
vector, re-circularized vector, and vector with insert (both
circular and linear). The concentration of all components is
usually about 50 ng/µL. Typically, the non-transforming
DNA will be in the majority, but it will not usually outcompete the transforming DNA. With 20 ng of total DNA
Donor DNA should not have detergent, phenol, alcohol,
PEG, or DNA binding protein in it. For electroporation,
donor DNA cannot have salt in it. Ligase and PEG strongly
inhibit transformation. A central problem in molecular
biology is that both ligase and PEG are components of
most ligation reactions. The best way to resolve this
problem is to precipitate the ligation mixture, dilute the
mixture three-fold and transform with 1 µL.
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Amount of DNA
10
Section 3: TRANSFORMATION
What is Important in a Transformation?
Storage and Handling of Competent Cells
Proper storage and handling of competent cells is one of
the most important factors affecting transformation.
Simply handling the cells with care will prevent loss of
efficiency.
Arrival
When the cells arrive in dry ice, they must be kept at
–70 °C to retain maximum efficiency. This can be achieved
by taking a few precautions. First, make sure the space is
ready in the –70 °C for the cells before unpacking them.
Once the space is ready, unpack the box at the freezer,
place the cells in the pre-selected space in the –70 °C, and
shut the door. There is no need to rush, but try to get the
cells into the –70 °C with as little delay as possible. Even
though the cells are frozen, they lose efficiency when they
do warm up even a little.
Storage
Although the cells are stored at –70 °C, they will be
subjected to temperature fluctuations constantly. When
the –70 °C is opened, warm air goes in and cold air goes
out. This is unavoidable, but the cells can be protected if
they are stored in a spot where they are not in the way
and are stored on a shelf that is not accessed very often.
between your fingers. DO NOT thaw the cells in a water
bath. By doing so, warming cannot be stopped when the
cells reach 0 °C. If the cells stay in an ice bucket for one
hour, that is acceptable. Beyond one hour, cells start losing
efficiency approximately two-fold each hour. If the cells are
left overnight, DO NOT use them! Use a new vial of cells.
Handling
Treat the cells gently. DO NOT pipet or vortex the cells. Mix
the cells by gently tapping on the tube.
Refreezing Cells
If a tube of cells is thawed and all of the cells are not
used, the remainder can be frozen. To refreeze, place the
tube in crushed dry ice, in a dry ice-ethanol bath (the best
option), buried in a bed of dry ice (the second best option)
or by itself on a metal shelf in the –70 °C for one hour
before placing it in the box. Efficiency will drop about twofold. If the tube is simply placed back into the box and
stored in the freezer, efficiency will drop five- to ten-fold.
Thawing the Cells
To thaw the cells, remove the vial from the –70 °C and thaw
on ice for 5 – 10 minutes. The cells must be directly on ice!
If time is an issue, the cells can be thawed by rolling the vial
Tips for Handling Competent Cells
1. Prepare space in the –70 °C freezer before
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unpacking the cells.
2. Keep the freezer door open for the shortest
amount of time possible.
3. Thaw the cells directly on ice.
4. Be gentle when mixing.
5. Refreeze properly.
11
Section 4: TRANSFORMATION PROTOCOLS — CLONING
Chemical Transformation
Chemical transformation is achieved by suspending the cells in an ice-cold buffer that contains calcium chloride
and other salts. Transformation occurs then the cells are warmed briefly. After transformation, the cells are
diluted into media to recover. Finally, the cells are plated onto media that selects for transformants. Although the
process is simple, there are important factors to remember when performing a chemical transformation.
What is Important in Chemical Transformation?
Purity of the DNA
Transformation frequency is affected by the purity of the
DNA used. For example, too much salt in the DNA when
performing electroporation can cause cells to explode.
Whether the DNA comes from a PCR reaction, ligation,
endonuclease digestion or other treatment, procedures to
further purify the DNA can be performed if needed.
The following procedures are recommended to remove the
impurities listed:
Impurity
Procedure
Proteins
Column purify the DNA or perform a
phenol extraction.
Detergents
Perform ethanol precipitation to purify
the DNA.
PEG
Column purify of perform either
precipitation or ethanol purification.
Amount of Ligation Mix
The most common mistake when performing a
transformation is to put too much ligation mix into the
transformation. The protocols suggest using less than 1 µl
of ligation. This amount is sufficient for any type of
transformation. Adding more lowers the number of
transformants. For chemically competent cells, the ligase
and PEG in the mix inhibits transformation. To get the
most transformants out of a ligation, there are two
options:
1. Precipitate the ligation and resuspend it in TE.
Incubating the DNA with the Cells on Ice
Incubating on ice for 30 minutes is required for chemically
competent cells. If the ice step is omitted and heat shock is
performed immediately, efficiency will drop ten-fold. If
incubated for only 15 minutes, efficiency will decrease threefold. Occasionally, this is a corner to cut if pressed for time and
maximum efficiency is not an issue.
Heat Shock
The heat shock works best in a thin-walled tube with a
42 °C water bath. A 45-second heat shock at 42 °C produces
the best results, but one minute at 37 °C works almost as well
(down two-fold). With Sigma-Aldrich Unipack cells, a 30second pulse at 37 °C in the tube provides the best results.
Recovery Time
The effect of the expression time depends on the plasmid and
strain. With pUC19 and GC5™, the efficiency is down ten-fold
if plated without any expression time at all. It is down sevenfold if plated after 15 minutes and down three-fold if plated
after 30 minutes. SOC medium provides two-fold better results
than LB medium for chemically competent cells.
Plates
Some plates provide better results than others, but there are
no magic plates. Plates less than six months old that are not
too dry will provide good results. Be especially cautious with
tetracycline plates. Tetracycline breaks down in light and
produces toxic products that kill everything but contaminants.
For best results, add the tetracycline to the plates when the
agar has cooled and is ready to pour. Discard the plates after
three months.
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2. Dilute the reaction three-fold in TE and use 1 µl per
50 µl competent cells.
12
Section 4: TRANSFORMATION PROTOCOLS — CLONING
Chemical Transformation
Chemical Transformation Protocol
Add plasmid DNA to cells.
Preparation:
1. Equilibrate a non-shaking water bath to 42 °C.
2. Prepare LB agar plates with the appropriate antibiotic. If
blue/white screening is desired, the plates should include
40 µg/ml X-Gal and 1 mM IPTG.
3. Agar plates should be placed in a 37 °C incubator for
30 minutes prior to plating.
4. Warm SOC to room temperature (20 – 25 °C).
Hold cells and plasmid on ice for 30 minutes.
Standard Procedure:
1. Remove the required tubes of cells from the -70 °C freezer,
including one extra for the control DNA if desired. Place
tubes immediately on wet ice so that only the cap is visible
above the ice. Allow the cells to thaw on ice for
approximately 5 minutes.
2. Visually examine the cells to ensure that they are thawed.
Gently tap the vial several times to resuspend cells.
3. (Optional) Add 1 µl pUC19 control DNA to one tube of cells.
Mix gently by tapping the tube. Return the cells to the ice.
Heat shock cells for 45 seconds at 42 ºC.
4. Add 1 – 50 ng of the ligation reaction or purified plasmid
DNA directly to cells. Mix as in step 3.
5. Incubate the cells on wet ice for 30 minutes.
6. Heat shock the cells by incubating the tubes in a
42 °C water bath for exactly 45 seconds.
7. Place the cells on ice immediately for at least
2 minutes.
8. Add 450 µl of room temperature SOC medium
into each tube containing the cell/DNA mixture.
Dilute E. coli transformed cells into SOC
and incubate for one hour.
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9. Incubate the cells at 37 °C with shaking
(225 – 250 rpm) for 1 hour.
10. Pipette 10 – 100 µl of each transformed cell suspension onto
LB agar plates containing selection antibiotic and evenly
distribute using a sterile spreader. Plates should be prewarmed to 37 °C for optimal colony growth. When
inoculating less than 25 µl of cell suspension, first pipette a
drop of SOC onto the plate and then add the cell suspension
to the SOC.
Note: The amount of transformation mixture to plate varies
with the efficiency of both the ligation and the competent
cells. When using the control DNA, add no more than 10 µl
into a drop of SOC on an LB agar plate containing 100 µg/ml
ampicillin or carbenicillin.
11. Incubate the plates at 37 °C overnight (12 – 16 hours).
Plate and select for resistance.
13
Section 4: TRANSFORMATION PROTOCOLS — CLONING
Electroporation
Electroporation of E. coli requires a high cell density and a non-ionic buffer. The same rules for storing and
handling chemically competent cells apply to electrocompetent cells. In electroporation, the competent cells
are thawed, mixed with donor DNA and placed in an electroporation chamber attached to an electroporation
device. The apparatus delivers a 5-millisecond pulse of about 1,900 volts. Efficiencies of 1010 transformants per
µg pUC19 DNA are expected from commercially prepared cells and efficiencies of 109 for home-made cells.
Conductivity of the Sample
This is the most important factor. The conductivity of the
sample should be as close to zero as possible. The number
one cause of exploding electrocompetent cells is putting
too much ligation mix into the transformation reaction.
Thawing the Cells
To thaw the cells, remove the vial from the –70 °C freezer
and thaw on ice for 5 – 10 minutes. The cells must be
directly on ice. If time is an issue, the cells can be thawed
by rolling the vial between your fingers. DO NOT thaw
cells in a water bath. By doing so, warming cannot be
stopped when the cells reach 0 °C. If the cells stay in an
ice bucket for one hour, that is acceptable. Beyond one
hour, cells start losing efficiency approximately two-fold
each hour. If the cells are left overnight, DO NOT use
them! Use a new vial of cells.
Incubating the Cells with DNA on Ice
It is not necessary to incubate the cells with DNA on ice.
The cells can be left on ice for one hour, after that,
efficiency decreases.
The Pulse
Instead of a heat shock, the cells are exposed to a very
short, intense electric field. The pulse has to be 4 – 5
milliseconds at a minimum field strength of 20 kV per cm
for Thunderbolt™ GC10™ cells. The field strength is usually
achieved with a voltage of 2.0 kV and a 0.1 cm cuvette.
With a 0.2 cm cuvette, it is impossible to reach this field
strength because most machines can only deliver 2.5 kV.
The length of the pulse is often achieved with a 25 uF
capacitor and a 200 Ω shunt resistor.
Note that there are two combinations of voltage and pulse
length that have proven to be efficient and practical: a
higher voltage with a shorter pulse length and a slightly
lower voltage with a longer pulse length. Both have been
used effectively, but one combination may prove to be
better under certain circumstances.
Shorter pulse: 2.5 kV, 100 Ω, 25 µF
Longer pulse: 2.0 kV, 200 Ω, 25 µF
Sigma-Aldrich offers two elecroporators: The
Electroporator, EC100 (catalog number Z375942) with AC
input 120V and the Electroporator, EC100 (catalog
number Z375950) with AC input 240V.
Recovery Time
The effect of the expression time depends on the plasmid
and the strain. Electroporation of Thunderbolt GC10 cells
with pUC19 only decreases two-fold in efficiency if plated
without any expression time at all.
Plates
Plates are not an issue. Since electrocompetent cells are so
concentrated, there can be an increased tendency to form
satellites when high cell densities are plated. Satellites are
untransformed cells that form small colonies in clusters
around real transformants. Satellites do not grow when
streaked on selective agar or when inoculated into
selective media.
Optimizing a New Strain
The best transformation uses pulses of 4 – 5 milliseconds,
although some protocols call for as much as a 10-millisecond
pulse or as little as 2. To optimize for a new strain, start with
a 4 – 5 millisecond pulse and vary the voltage so that the
field strength varies from 15 kV/cm to as high as possible
without exploding the cells (20 – 25 kV/cm). The pulse can
be varied from 2 ms to 10 ms by changing the value of the
shunt resistor. Try combinations. If the cells always explode,
the problem may not be the pulse and voltage combination
used, but may be due to too much salt in the cells. Try
washing them again before electroporating.
Salts and Buffers Inhibit Electroporation
Experimental DNA should be in a low ionic strength buffer
such as TE. Samples containing too much salt will result in
arching at high voltage that could harm the sample and/or
the equipment. Ligation reactions should be diluted 5-fold
in TE buffer prior to transformation. Use 1 µL of the diluted
ligation reaction per 40 µL of electrocompetent cells.
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What’s Important in Electroporation?
14
Section 4: TRANSFORMATION PROTOCOLS — CLONING
Electroporation
Electroporation Protocol
(Using Thunderbolt™ GC10™ cells)
Add plasmid DNA to E. coli.
Preparation
1. Place the electroporation chamber on ice.
2. Prepare LB agar plates with the appropriate antibiotic.
If blue/white screening is desired, the plates should
include 40 µg/mL X-gal and 1 mM IPTG.
3. Agar plates should be placed in a 37 °C incubator
30 minutes prior to plating.
Place the DNA and cells into a chilled cuvette.
4. Warm SOC medium to room temperature (20 – 25 ºC).
Procedure
1. Remove the cells from the -70 °C freezer and place
directly in ice.
2. Place 1 mm standard cuvettes and autoclaved
microcentrifuge tubes on ice, one per transformation.
3. Place 960 µL SOC medium in culture tubes, one per
transformation.
Place the cuvette into the electric field and apply charge.
4. Add the DNA (or 1 µl of control DNA diluted 5-fold)
to the microcentrifuge tubes on ice.
5. Gently mix cells by tapping the tube.
6. Transfer 40 µl of the cells into chilled tubes
containing DNA.
7. Pipet 40 µl of the DNA/cells mixture into a chilled
1 mm cuvette.
8. Electroporate at a field strength of 20 kV/cm for 6 ms.
+
-
Dilute E. coli transformed cells into SOC medium
and incubate for one hour.
9. Remove cells from the cuvette and place in culture
tubes containing SOC medium.
10. Incubate at 37 °C for one hour with shaking
(225 – 250 rpm).
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11. Spread on LB agar plates containing appropriate
antibiotic (e.g., 100 µg/mL ampicillin for control
pUC19).
12. Incubate the plates at 37 °C overnight (12 – 16 hrs).
Plate and select for resistance.
15
Section 4: TRANSFORMATION PROTOCOLS — CLONING
FAQs on Cloning
What are the optimal settings for electroporation?
There are two combinations of voltage and pulse length
that have proven to be efficient and practical: a higher
voltage with a shorter pulse length and a slightly lower
voltage with a longer pulse length. Both have been used
effectively, but one combination may prove to be better in
your laboratory.
Shorter pulse: 2.5 kV, 100 Ω, 25 mF
Longer pulse: 2.0 kV, 200 Ω, 25 mF
Why didn’t I get any transformants (chemical
transformation)?
There are multiple reasons that this could occur:
• The cells might not have been competent. Try the
control DNA to make sure the cells are competent.
• The ligation might have failed. Does this donor work
on any cells?
• Was the correct selection used for the plasmid?
• Were the cells concentrated by centrifugation?
If so, were the cells handled gently? Try plating
0.1 mL before concentrating the rest of the cells.
Why do I have a lot of little colonies around
the big colonies?
These are satellite colonies. They are not transformants.
Incubate the plates for less time, use more antibiotic, or
use fresh plates to get rid of them. If 0.1 mL of an
electroporation was plated, there will most likely be
satellites. Ignore them, they will not grow overnight if
using selection.
Why does my plate have colonies of all sizes?
The selection is off. There is either too much or too little
antibiotic. To determine the problem, streak the cells that
should grow on the selection and cells that should not. If
the cells that should grow are struggling, there is too
much antibiotic and transformants are being lost. Try half
as much. If the cells that should not grow are growing
where the steak is heaviest, there is not enough
antibiotic. If there is not enough antibiotic,
“breakthrough” of non-transformed cells that are
mutants to a low level of resistance will appear. Try using
twice as much antibiotic.
Why do my plates look like one giant colony?
Try the suggestions listed for chemical transformation.
If the control did not work either, make sure that
everything is hooked up correctly. If it is, do a test pulse:
1. Add 40 µL of LB or SOC to a used cuvette.
It should arc.
2. If it does not arc, there is no connection or the
machine is dead.
• Have the cuvette chamber and the cables tested
to make sure they conduct electricity.
• If they are OK, the electroporation machine may
be dead. Call the manufacturer.
Why did my cells explode when I pulsed them
(electroporation)?
• Too much DNA was added to the reaction. Try
adding less DNA or ethanol precipitating the DNA.
• Check the settings. Use 200 Ω. 2.0 kV, 25 µF
capacitor. The lower voltage is less likely to
pop the cells.
• The cells may have too much salt in them. If you
made them yourself, wash them one more time.
The first thing to determine is if there is antibiotic on the
plate. If not, don’t forget it next time. Another possibility
is that the plate was wet. If so, these cells are swimmers.
When I use the control and calculate transformation
efficiency, I get a number that is 2 – 4 times as
high as the specification. Is this OK?
The specification is a minimum. This is good!
I left my cells in the ice bucket overnight. Are
they still OK?
No. Do not use them unless you are desperate.
My freezer died, but the temperature only went
to –50 °C before I was able to transfer the cells to
a different –70 °C freezer. Can I still use them?
Yes, but expect a two- to five-fold loss in efficiency.
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Why didn’t I get any transformants
(electroporation)?
16
Section 5: TRANSFORMATION PROTOCOLS — EXPRESSION
The procedures to transform are the same whether cloning or expressing. The important points to
consider before beginning a transformation should still be followed. Additionally, there are steps to take
when working with BL21 for successful transformation.
Making a Stock Culture
The first thing to do when working with BL21s is to make
a stock culture. This ensures that the clone does not
change and that each expression run gives optimal
performance.
1. Transform the BL21 strain to be used with the plasmid.
2. Pick a single transformant colony from a fresh plate
into 30 mL of LB + ampicillin (+ chloramphenicol as
well for pLysS or pLysE). A small amount will do.
3. Grow overnight. Room temperature is best. Turn the
heater off on the shaker. Don’t worry, the cells will
grow. Many people cannot just shut off the heat on a
shared lab shaker. In that case, grow the cells at 30 ºC.
If this is not possible and they have to grow at 37 ºC,
make three cultures: one at full strength, the second a
10-fold dilution of the first flask, the third a 10-fold
dilution of that. Use the flask that grew from the most
diluted inoculum (this is the one that spent the least
amount of time at stationary phase).
4. In the morning, dilute 10 mL of overnight culture
with 10 mL of LB-20% glycerol.
5. Distribute 1mL each into 1.2 mL cryotubes (5 – 20
tubes). Freeze and store at -70 ºC. As long as they
stay at -70 ºC, they will be unchanged.
When down to the last tube, make a new stock culture.
Using the original plasmid, make it the same way. Just
subculture from the stock culture. Do a new test expression
first to make sure that the strain has not lost viability. DO
NOT use a culture that has been thawed more than once.
To subculture from a master tube without thawing it,
remove the tube and place it on dry ice. Open it and
scrape some material from the top with an inoculation
loop or a toothpick. Inoculate in 1 ml of culture media
with the scraping. Replace the tube in the freezer. DO
NOT store the cultures as stabs, on plates, or in a tube in
the refrigerator. When this is done, most of the cells die.
Often, the cells that do not die are the cells that will not
make the protein of interest anymore.
DO NOT make stock cultures from cells that have
been induced.
Important: When making a stock culture, remember that
in log growth, the T7 system is repressed and all cells are
more or less competitive. In stationary phase, cells are
stressed and many will die. Cells with the least amount
of stress will survive. Therefore, never make a stock culture
from cells that have been induced.
6. Each time an expression is done, thaw out a stock
culture and use that to start the culture.
Tips for Transformation:
What is Important
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1. Purity of the DNA
2. Amount of Ligation Mix
3. Incubating the DNA with Cells on Ice
4. Heat Shock or the Pulse
5. Recovery Time
6. Plates
Please see p. 12 and p. 14 for detailed explanations.
17
Section 5: TRANSFORMATION PROTOCOLS — EXPRESSION
General Expression Protocol
• Does the clone really make the desired protein?
Separation of Insoluble and Soluble Protein
1. Dilute the samples to 2 mg/mL protein.
2. Sonicate the samples to disrupt the cells.
3. Remove 10 mL and electrophorese.
This is the total protein.
• How much protein is made?
• Is the protein soluble or insoluble?
Growth and Sampling
The following is a generic protocol that lacks some detail
as many different types of media and methods are used.
Cells in LB grow to a maximum of about 3 OD with good
aeration. In rich media such as Terrific Broth, culture OD
can reach 20 with excellent aeration. Minimal media can
give results as low as 0.5 OD or as high as 20 OD,
depending on conditions.
1. Dilute 1 mL of stock culture into 100 mL of media +
ampicillin (+ chloramphenicol as well for pLysS or
pLysE) in a 500 mL baffled flask.
[Amp = 100 µL/mL, Cam = 25 µL/mL]
2. Grow the cells to 0.5 OD at 37 °C. This takes 2 – 3
hours. During this time, label five 15 mL conical
centrifuge tubes as follows: 0, 1, 2, 3, O/N.
3. Harvest 10 mL of the uninduced (0 hours) sample.
Spin the tube at 4,000 rpm for 20 minutes. Pour off
the supernatant. Freeze the pellets.
4. Add 1 mL of 100 mM IPTG to the culture
[final concentration will be 10 mM].
5. Measure the OD of the cells for each of the next 3
hours. Harvest 10 mL samples of the culture at each
time: 1 hour, 2 hours and 3 hours after induction.
Store the pellets at –20 °C.
6. Continue to express the cells overnight.
7. The next morning, harvest 10 mL of the cells.
Note the time.
4. Spin at 13,000 rpm for 5 minutes.
5. Remove 10 mL and electrophorese.
This is the soluble fraction.
Protein Determination
The approximate amount of total protein (in mg) that can
be expected in each sample depends on the OD of the
culture when it was sampled, as follows:
OD of culture when sampled:
0.5
1
2
3
4
5
7.5
10
15
20
Approximate concentration of protein in the 1 mL
resuspension of the sample pellet (mg/mL):
0.25 0.5
1
1.5
2
2.5
3.8
5
7.5
10
Resuspend the pellets in 1 mL TE. Measure the total
protein in each of the 1 mL samples using Bradford
(Catalog Number B6916), BCA (Catalog Number BCA1), or
equivalent with a standard curve. It is important to do this,
as the gel analysis is much easier if each lane has the same
amount of protein.
Gel Electrophoresis
Load the same amount of protein in each lane of the gel.
Analyze the samples by gel electrophoresis, looking at all of
the samples: 0 (uninduced), 1, 2, 3, and O/N hours of
induction. It is recommended to compare soluble protein and
total protein. Insoluble protein is the total minus the soluble.
The amount of protein in each sample depends on the
amount of cells. The amount of cells in each sample depends
on the clone and the media being used. Measure the amount
of protein and load the same amount of protein in each lane
of the gel.
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Test Expression
The key to a successful gene expression is to do a test
expression first to determine the following:
18
Section 5: TRANSFORMATION PROTOCOLS — EXPRESSION
General Expression Protocol
The test expression experiment can be recorded in any way
you like. A form such as this, or a derivative can be used:
Time – time of day time points were taken.
Date:
________________
Host Strain:
________________
Plasmid:
________________
Medium:
________________
Antibiotics:
Ampicillin _______
Time Post-Inoc – hours after inoculation. The target times are
shown. The actual times should be recorded next to the target
times.
Chloramphenicol _______
Other_______
Time
_______
Time post-inoc.
0
OD
___
Vol. Assayed
__________
Reading Protein/mL
_______ _________
Total Protein
__________
_______
1
___
__________
_______ _________
__________
_______
2
___
__________
_______ _________
__________
_______
3
___
__________
_______ _________
__________
_______
O/N
___
__________
_______ _________
__________
Vol. Assayed
Reading Protein/mL
Standard protein
0
_______
OD – OD of the cell culture when it was sampled. The
wavelength is not crucial: 550, 590, 595, 600 nm all give
similar results.
Vol. Assayed – the volume of resuspension that was
assayed.This gives an estimate of the concentration of
protein in the sample.
Reading – the reading of the protein assay (e.g. OD562 for
BCA or OD595 for Bradford).
0
__________
_______ _________
__________
_______ _________
__________
_______ _________
__________
_______ _________
__________
_______ _________
__________
_______ _________
__________
_______ _________
Protein/mL – with the standard protein, this is calculated
by the concentration of the standard, the volume assayed,
and the volume of the assay. For the cell samples, the
protein concentration/mL is determined by the standard
curve, the sample reading, and the conversion factor
determined by the results.
Total protein – “resuspended volume” x “protein/mL”
Analysis of Test Expression Results
There are many possible outcomes to the test expression
experiment. Potential results include:
• Cells are not inhibited by induction and produce a lot of
soluble protein. This is ideal. Scaling up is recommended.
www.sigma-aldrich.com
• Cells are not inhibited by induction, but do not make
very much protein. This is not the ideal result; however,
an amino terminal fusion can be made with a protein
that E. coli does express well, such as thioredoxin. If the
fusion protein is made, initiation of translation of the
protein is inefficient. If using E. coli, stay with the
amino fusion.
• Cells are inhibited by induction and make a lot of protein.
This is good. Induce at the highest OD possible, which
will depend on the media being used and aeration. Induce
at 1/3 of the final OD obtained when not inducing.
• Cells are inhibited by induction and do not make very
much protein. This is not good. Induce at the highest OD
possible, which will depend on the media being used
and aeration. Induce at 1/3 the final OD obtained when
not inducing. Whatever protein is being produced is
killing off the cells. This is the best that can be achieved
in this system.
19
Section 5: TRANSFORMATION PROTOCOLS — EXPRESSION
FAQs on Expression
The stringy material is dead cells. The survivors will not
make as much of the protein. Retransform BL21 with the
original plasmid and make stock cultures. Never store
BL21 on a plate as this discriminates against cells that are
making the protein. Storage on a plate “favors” the
wrong cells.
I’m working with BL21(DE3) pLysS and I’m getting
a lot of background expression. Why?
Is selection done for pLysS as well as the plasmid? This
should be done by placing chloramphenicol (Cm) in
selective plates and growth media. If selecting with Cm
and still getting a lot of background, then the construct is
super hot. If this is a problem, switch to a promoter
system that has better control, such as PL. Some
researches use BL21(DE3)pLysE because it produces even
more inhibitor of background T7 expression. This strain
can be tricky to use and sometimes grows poorly, which
could be due to increased background expression of T7
lysozyme from the tet promoter.
I’m working with BL21(DE3)pLysS and getting
different sized colonies. Why?
Is there chloramphenicol in the plates? If not, big colonies
may be ones in which the pLysS plasmid has been lost,
because it inhibits growth. On the other hand, the big
colonies might be the one WITH pLysS because the
background expression of your gene is killing the cells in
the absence of pLysS. Streak the big colonies and the little
colonies on chloramphenicol to see which is occurring.
I left my cells in the ice bucket overnight.
Can I still use them?
Not unless it is a desperate situation. Get a new vial
of cells.
When I induce my cells, they stop growing. After
three hours, there is stringy stuff in the flask.
After incubating overnight, there are not a lot of
cells, but I can hardly see my protein. Why?
If the OD level is off when the cells are induced, this will
happen. If the OD drops, that means that the induced
protein is lethal. Overnight, something grows and it is
probably something that has killed the protein gene.
What can be done now is to grow to 1 OD before
induction, and harvest in 1 – 3 hours.
When I do the control and calculate
transformation efficiency, I get a number that is
2 – 4 times as high as the specification. Is this OK?
The specification is a minimum. We are getting BL21s
at 108 quite often.
I checked the plasmid in my BL21 transformant
and the digest looks like it is degraded. What
could be wrong?
BL21 derivatives have an endonuclease that degrades all DNA.
Extract the plasmid prep with an equal volume of phenol, and
then ethanol precipitate. The yield should be more than 50%
and the resulting plasmid prep should be fine.
Can I clone in BL21?
Yes. Success depends on how efficient the cloning is.
If using a fancy cloning method with high yield and
low background, then transformation straight into BL21
can be done to save time. If cutting, pasting and
screening, it is better to use a cloning strain (like GC5™)
until the desired result is achieved before putting the
plasmid into BL21.
My freezer died , but the temperature only went
to –50 ˚C before I was able to transfer the cells to
a different –70 ˚C freezer. Can I still use them?
Yes, but expect a two- to five-fold decrease in efficiency.
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I store my cultures on plates that I keep in my
refrigerator. When I inoculate media with these
cultures, the cells are stringy and take a long time
to grow. Why?
20
Appendix 1: GENOTYPES
Genotypes
Blue/white screening: pUC19 and similar plasmids code
for β-galactosidase (lacZ), which cleaves X-gal and turns
colonies blue on X-gal plates. Inserts cloned into the
plasmid disrupt the β-galactosidase gene resulting in white
colonies. The plasmids only code for a small part of the βgalactosidase gene (the α peptide) and the chromosome
codes for the rest. Both parts are required for activity.
Since the plasmid is complementing the chromosomal
mutation, this effect is called “α complementation.”
Cytosine Methylation deficient (dcm)
It is unknown why E. coli K12 methylates certain sites
(CCAGG and CCTGG). E. coli B does not do this, therefore
BL21 does not do this.
Endonuclease Deficient (endA)
E. coli has a powerful endonuclease on the outside of the
cell that degrades any type of DNA. EndA mutants do not
have this capability. The endA endonuclease has little or no
effect on transformation efficiencies, but can have a great
effect on the quality of plasmid DNA preparation. If
plasmid DNA preps degrade when placed in magnesiumcontaining bugs, it is usually because the DNA was made
from an endA bug. To prevent DNA degradation in
plasmid preps, choose an endA– host.
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Galactose Metabolism deficient (gal)
This mutation prevents BL21 from using galactose as a
carbon source.
lac Promoter Control (lacIQ)
It is important when making plasmid constructs to keep the
expression promoter off until ready to turn it on. High-level
expression of many genes is bad for the host. When this
happens, mutants that do not express the gene at a high
level grow faster and take over the culture. Some expression
systems use the lac, tac, or trc promoters to express cloned
genes on high-copy plasmids, but do not have the cognate
repressors on the expression plasmid. Under these
circumstances, there is not enough lac repressor to keep the
promoters off. A mutant that produces more lac repressor
(lacIQ) can repress lac, tac, and trc promoters until IPTG is
added to induce them.
lon protease deficient (lon)
The parental E.coli B strain is a lon mutant, and therefore
BL21s are lon mutants. The lon protein serves two functions
in E. coli: it degrades misfolded (abnormal) proteins and it
degrades normal proteins. This is an intracellular process,
which includes degradation of the protein that you want to
express. The lon protease degrades the protein before the
cells are even lysed. To avoid this, expression strains are lon –.
Methyl Restriction Deficient (mcrA, mcrB, mrr)
E. coli has a system of enzymes that degrade DNA if it is
methylated at the “wrong” sites. Genomic DNA from
eukaryotic sources is methylated at all of the wrong sites as
viewed by E. coli. When cloning genomic DNA from
eukaryotic cells, it is essential to use a host that is deficient
in all three of these methyl restriction systems. When
cloning PCR fragments, cDNA, or fragments from previously
made clones, there is no methyl restriction and it is not
necessary to use a methyl restriction deficient host. When
cloning genomic DNA, the cloning hosts should be mcr –.
Outer Membrane Protease deficient (ompT)
ompT is a protease that E. coli makes which sits on the
outer surface to degrade extracellular proteins. It degrades
the protein after the cells are lysed. Those that prefer intact
protein demand that the E. coli proteases be eliminated, but
this is not entirely possible. By eliminating ompT and lon,
BL21s are able to maintain an optimal balance.
pLysS and pLysE
The pLysS and pLysE plasmids express T7 lysozyme, a natural
inhibitor of T7 polymerase, allowing for improved
transcriptional control and reduction of "leaky" expression.
The pLysE plasmid expresses T7 lysozyme at higher levels
than the pLysS plasmid conferring a greater level of control
over the T7 polymerase. This is usually required only when
the recombinant protein to be expressed may be toxic to the
cell. Both the pLysS plasmid and the pLysE plasmid render
the cells resistant to chloramphenicol (CmR) and also contain
the p15A origin, which allows compatibility with plasmids
containing ColE1 or pMB1 (derivative of pBR322) origin.
21
Appendix 1: GENOTYPES
Genotypes
Phage Resistance (fhuA, tonA, T1R)
T1 phage and related phages kill E. coli. It is possible to
have T1 phage infection. Unlike other phage, T1 is
resistant to drying and is almost impossible to eliminate.
The T1 resistance marker protects clones and cells with T1
resistance are fast becoming standards in the lab.
Recombination Deficient (recA)
E. coli has a repair system that will recombine homologous
sequences. There is concern that recombination can cause
plasmids to rearrange or to delete insertions. As a result,
cloning strains are generally recA mutants. recA strains
have the advantage of having a simple plasmid profile.
recA+ strains have dimers, trimers and their nicked
relatives. To prevent almost all homologous recombination,
choose a recA– host.
Genomic clones often have duplicated regions, but
they are generally short, tandem duplications. These
duplications are unstable, but this is not due to recA
function. If the length of the duplicated sequence is less
than 200 bp, recA has no effect.
Single Strand Ability (F+ or F′)
F is a huge plasmid (99 kb) that is naturally found in E. coli
K12. There are derivatives of the F plasmid that also
contain chromosomal DNA. These F derivatives with a bit
of chromosomal DNA are called F′ plasmid. E. coli with
the F (or F′) plasmid make special surface features that
allow them to be infected with M12 and similar phage.
This property is useful if one wants to make singlestranded DNA or generate phage display libraries.
T7 polymerase (λDE3 with lacUV5-T7 gene 1)
When people say BL21, they usually mean BL21(DE3), which is
a derivative of BL21 that has the T7 RNA polymerase gene
under the control of the lacUV5 promoter. The lacUV5
promoter is a mutant lac promoter that is stronger than a wild
type lac promoter. The whole arrangement is on a λ phage
genome, and this particular version of the λ phage is called
λDE3. λDE3 is inserted into the chromosome of BL21 to make
BL21(DE3). There are other markers on this λDE3 genome
(lacI, ind1, sam7, nin9), but none will affect expression.
The recA repair system is also useful to E. coli. Disabling it
will cause the cells to grow slower and they are generally
less healthy. For this reason, some expression strains are
not recA mutants.
For more information on genotypes,
visit sigma-aldrich.com/competentcells
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Restriction Deficient (hsdRk–)
Most lab strains are E. coli K12 derived. K12 strains
methylate their DNA at K12 sites (AAC(N6)GTGC and
GCAC(N6)GTT). In K12 strains, DNA that is not methylated
at these sites is degraded by a restriction enzyme. Many, but
not all, cloning strains of E. coli are mutated in the gene that
codes for this restriction enzyme. BL21 (expression strain)
does not methylate, nor does it restrict unmethylated DNA.
To avoid restriction problems, all hosts should be hsdR –.
22
Appendix 2: SUPPORTING PRODUCTS
Supporting Products
Cat. No.
Description
Ampicillin
A2804
A0797
A0166
A5354
Ampicillin Sodium Salt-powder
Ampicillin Sodium Salt-powder, cell culture tested
Ampicillin Sodium Salt-powder, cell culture tested, minimum 98% (titration)
Ampicillin Ready-Made Solution
Carbenicillin
C9231
C1613
Carbenicillin Disodium Salt
Carbenicillin Ready-Made Solution
Electroporator, EC100
Z375942
Z375950
AC Input 120V
AC Input 240V
IPTG (Isopropyl β-D-1-thiogalactopyranoside)
I6758
I1284
IPTG
IPTG Ready-Made
LB Agar
L2897
L7533
C4478
LB Agar Powder
LB Agar EZMix Powder
S-Gal™/LB Agar Blend
LB Mediums
L3022
L7658
L2542
LB Broth Powder
LB Broth EZMix™ Powder
LB Broth Liquid
Pre-Poured Agar Plates
L5667
L5542
LB Agar Ampicillin - 100 (10 plates)
LB Agar (10 plates)
Protein Determination Reagents
BCA1
B6916
Bicinchoninic Acid (BCA) Kit
Bradford Reagent
SOC Medium
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S1797
SOC Medium
Terrific Broth
T0918
T9179
T5574
Terrific Broth, Modified Powder
Terrific Broth, Modified EZMix Powder
Terrific Broth Liquid
X-gal
B9146
B6024
X-gal, minimum 98%
X-gal, tablet
EZMix™ and S-Gal™ are trademarks of Sigma-Aldrich.
23
ACKNOWLEDGEMENTS AND GENERAL REFERENCES
Acknowledgements
We would like to thank GeneChoice, Inc. for their support and content in the Competent Cell Compendium: Tools and Tips for
Successful Transformation.
General Reference
High Efficiency Transformation of E. coli Grown at 37 ˚C
Hanahan, D. (1983). Studies on transformation of Escherichia coli with
plasmids. J. Mol. Biol. 166: 557-580.
General Review and Methods of Preparing and Using Competent Cells
Hanahan, D., J. Jessee and F. Bloom (1991). Plasmid transformation of
Escherichia coli and other bacteria. Method in Enzymology 204:
63-113.
General Review of Transformation of Bacteria
Smith, H.O., D.B. Danner and R.A. Reich (1981). Genetic Transformation.
Annual Review of Biochemistry 50: 41-68.
High Efficiency Transformation of E. coli Grown at 18 ˚C
Inoue et al. (1990). Gene 96: 23-28
Electroporation of E. coli
1. Calvin, N.M. and P.C. Hanawalt (1988). J. Bacteriology 170: 27962801.
2. Dower, W.J. and C.W. Ragscale (1988). Nucleic Acids Research 16:
6127-6145.
Practical Aspects of E. coli Electrporation
1. Smith, M.D., J. Jessee, T. Landers and J. Jordon (1990). High Efficiency
Bacterial Electroporation: 1 x 1010 E. coli Transformants/mg. Focus
12: 38-41.
2. Li, S.J., T.A. Landers and M.D. Smith. Electroporation of plasmids into
plasmid-containing Escherichia coli. Focus 12: 72-75.
Everything about Molecular Cloning — Transformations, too
1. Sambrook, J., E. Fritsch and T. Maniatis (1989). Molecular Cloning: A
Laboratory Manual. Second Edition. Cold Spring Harbor Press,
Cold Spring Harbor, NY.
2. Sambrook, J. and D. Russell (2001). Molecular Cloning: A Laboratory
Manual. Third Edition. Cold Spring Harbor Press, Cold Spring
Harbor, NY.
Original Description of E. coli
Lim, B.A., E.T. Dimalanta, K.D. Potamousis, J. Apodaca, T.S. Anantharaman and E.M. Witkin (1946). Inherited differences in sensitivity to
radiation in Escherichia coli. Proc. Natl. Acad. Sci. USA 32: 59-68.
Derivation of BL21
Wood, W.B. (1966). Host specificity of DNA produced by Escherichia
coli: bacterial mutations affecting the restriction and modification
of DNA. J. Mol. Biol. 16: 118-133.
Original Decription of the T7 System
Studier, F.W. and B.A. Moffatt (1986). Use of bacteriophage T7 RNA
polymerase to direct selective high-level expression of cloned
genes. J. Mol. Biol. 189: 113-130.
pLysS and pLysE
Moffatt, B.A. and F.W. Studier (1987). T7 lysozyme inhibits transcription
by T7 RNA polymerase. Cell 49: 221-227.
Excellent Reviews on Gene Expression in E. coli
1. Baneyx, F. (1999). Recombinang protein expression in Escherichia
coli. Current Opinion in Biotechnology 10: 411-421.
2. Makrides, S.C. (1996). Strategies for achieving high-level expression
of genes in Escherichia coli. Microbiol. Rev. 60: 512-538.
3. Summers, D. (1998). Timing, self-control and a sense of direction are
the secrets of multicopy plasmid stability. Mol. Microbiol. 29:
1137-1145.
4. Goldstein, M.A. and R.H. Doi (1995). Prokaryotic promoters in
biotechnology. Biotechnol. Annu. Rev. 1: 105-128.
Rare Codons
Zhan, K. (1996). Overexpression of an mRNA dependent on rare codons
inhibits protein synthesis and cell growth. J. Bacteriol. 178:
2926-2933.
Fusion Proteins
To a modified DsbA
Zhang, Y., D.R. Olsen, K.B. Nguyen, P.S. Olson, E.T. Rhodes and D.
Mascarenhas (1998). Expression of eukaryotic proteins in soluble
form in Escherichia coli. Protein Expr. Purif. 12: 159-165.
To a phage protein
Forrer, P. and R. Jaussi (1998). High-level expression of soluble heterologous proteins in the cytoplasm of Escherichia coli by fusion to the
bacteriophage lambda head protein. D. Gene 224: 45-52.
To maltose binding protein
Kapust, R.B. and D.S. Waugh (1999). Escherichia coli maltose-binding
protein is uncommonly effective at promoting the solubility of
polypeptides to which it is fused. Protein Sci. 8: 1668-1674.
To anything, but the order of fusion matters
Sachdev, D. and J.M. Chirgwin (1998). Order of fusions between bacterial and mammalian proteins can determine solubilitye in
Escherichia coli. Biochem. Biophys. Res. Commun. 244: 933-937.
Proteases
Gottesman, S. (1996). Proteases and their targets in Escherichia coli.
Annu. Rev. Genet. 30: 465-506.
www.sigma-aldrich.com
Original Papers on Chemical Transformation of E. coli
1. Mandel, M. and A. Higa (1970). Calcium-dependent bacteriophage
DNA invection. J. Mol. Bio. 53: 159-162.
2. Cohen, S.N., A.C.Y. Chang and L. Hsu (1972). Nonchromosomal
antibiotic resistance in bacteria: genetic transformation of
Escherichia coli by R-factor DNA. Proc. Natl. Acad. Sci. USA 69:
2110-2114.
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