pET System Vectors and Hosts Instruction Manual

pET System Vectors and Hosts

INSTRUCTION MANUAL

Catalog #211521, #211523, #211621, and #211623

Revision A

For In Vitro Use Only

211521-12

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pET System Vectors and Hosts

CONTENTS

Materials Provided...................................................................................................................... 1

Storage Conditions...................................................................................................................... 1

Additional Materials Required .................................................................................................. 2

Academic and Nonprofit Laboratory Assurance Letter.......................................................... 2

Introduction................................................................................................................................. 3

pET Expression Vectors ................................................................................................ 3

pET-3 Vector Map......................................................................................................... 5

pET-11 Vector Map....................................................................................................... 6

Bacterial Strains ............................................................................................................ 7

Bacteriophage CE6........................................................................................................ 8

Cloning Protocol.......................................................................................................................... 9

Preparing the Vectors .................................................................................................... 9

Ligating the Insert........................................................................................................ 10

Transformation Protocol .......................................................................................................... 11

Transformation of the BL21-Gold Expression Strains................................................ 11

Transformation Summary for the pUC18 Control Plasmid......................................... 12

Expression Protocols................................................................................................................. 12

Induction of Target Protein Using IPTG ..................................................................... 12

Induction of Target Protein by Infection with Lambda CE6....................................... 13

Growth and Maintenance of High-Titer Bacteriophage Lambda CE6 Stocks ............ 14

Phage Amplification.................................................................................................... 14

Induction of Target Protein by Infection with Lambda CE6....................................... 14

Troubleshooting ........................................................................................................................ 16

Preparation of Media and Reagents........................................................................................ 17

References .................................................................................................................................. 19

Endnotes..................................................................................................................................... 19

MSDS Information.................................................................................................................... 19

Quick-Reference Protocol ........................................................................................................22

pET System* Vectors and Hosts

ATERIALS PROVIDED

M

Kit Quantity Catalog # Storage

pET expression systems

pET 3 vector series: 3a, b, c and d DNA

BL21-Gold(DE3) competent cells 10 × 0.1-ml –80°C

BL21-Gold(DE3)pLysS competent cells

pUC18 control plasmid (0.1 ng/μl in TE buffer)

pET 11 vector series: 11a, b, c and d DNA

BL21-Gold(DE3) competent cells 10 × 0.1-ml –80°C

BL21-Gold(DE3)pLysS competent cells

pUC18 control plasmid (0.1 ng/μl in TE buffer)

pET vectors

pET 3 vector series: 3a, b, c and d DNA

pET 11 vector series: 11a, b, c and d DNA

a

See Table I and Figure 1.

b

See Table I and Figure 2.

c

The pET 3a, b, c and pET 11a, b, c plasmids have one base pair shift in the BamH I site, from a to b and b to c.

d

The pET 3d and 11d plasmids have an Nco I cloning site, which is not present in a, b and c.

e

The pET 11 series has the lac operator and lacIq sequence for tighter expression control (not in the pET 3 series).

a,c,d

Four 20-μg tubes of

cesium chloride-banded,
supercoiled plasmid DNA

10 × 0.1-ml

2 ×10 μl

b,c,d,e

Four 20-μg tubes of cesium

chloride-banded,
supercoiled plasmid DNA

10 × 0.1-ml

2 ×10 μl

a,c,d

Four 20-μg tubes

containing cesium
chloride-banded,
supercoiled plasmid DNA

b,c,d,e

Four 20-μg tubes

containing cesium
chloride-banded,
supercoiled plasmid DNA

#211621

–20°C

–80°C

–20°C

#211623

–20°C

–80°C

–20°C

#211521 –20°C

#211523 –20°C

STORAGE CONDITIONS

Vectors: −20°C
Competent Cells: −80°C

* The pET system is covered by U.S. Patent No. 4,952,496. A nondistribution agreement accompanies the products.
Commercial customers must obtain a license agreement from Associated Universities before purchase.

Revision A © Agilent Technologies, Inc. 2008.

pET System Vectors and Hosts 1

ADDITIONAL MATERIALS REQUIRED

IPTG
β-Mercaptoethanol and Falcon® 2059 polypropylene tubes

calf intestinal alkaline phosphatase
T4 ligase

ACADEMIC AND NONPROFIT LABORATORY ASSURANCE LETTER

The T7 expression system is based on technology developed at Brookhaven National Laboratory
under contract with the U.S. Department of Energy and is protected by U.S. patents assigned to
Brookhaven Science Associates (BSA). BSA will grant a nonexclusive license for use of this
technology, including the enclosed materials, based on the following assurances:

1. These materials are to be used for noncommercial research purposes only. A separate license is
required for any commercial use, including the use of these materials for research purposes or
production purposes by any commercial entity. Information about commercial licenses may be
obtained from the Office of Intellectual Property and Industrial Partnerships, Brookhaven National
Laboratory, Bldg. 475D, Upton, New York, 11973 [telephone [phone]].

2. No materials that contain the cloned copy of T7 gene 1, the gene for T7 RNA polymerase, may be
distributed further to third parties outside of your laboratory, unless the recipient receives a copy of
this license and agrees to be bound by its terms. This limitation applies to strains BL21-Gold(DE3)
and BL21-Gold(DE3)pLysS included in this kit and any derivatives you may make of them.

You may refuse this license by returning the enclosed materials unused. By keeping or using the
enclosed materials, you agree to be bound by the terms of this license.

Commercial Entities Outside of the US

The T7 expression system is based on technology developed at Brookhaven National Laboratory
under contract with the U.S. Department of Energy and is protected by U.S. Patents assigned to
Brookhaven Science Associates (BSA). To protect its patent properties BSA requires commercial
entities doing business in the United States, its Territories or Possessions to obtain a license to
practice the technology. This applies for in-house research use of the T7 system as well as
commercial manufacturing using the system. Commercial entities outside the U.S. that are doing
business in the U.S., must also obtain a license in advance of purchasing T7 products. Commercial
entities outside the U.S. that are using the T7 system solely for in-house research need not obtain a
license if they do no business in the United States. However all customers, whether in the U.S. or
outside the U.S. must agree to the terms and conditions in the Assurance Letter which accompanies
the T7 products. Specifically, no materials that contain the cloned copy of T7 gene 1, the gene for
T7 RNA polymerase, may be distributed further to third parties outside of your laboratory, unless the
recipient receives a copy of the assurance letter and agrees to be bound by its terms. This limitation
applies to strains BL21-Gold(DE3) and BL21-Gold(DE3)pLysS included in this kit and any
derivatives you may make of them.

To obtain information about licensing, please contact the Office of Intellectual Property and
Industrial Partnerships, Brookhaven National Laboratory, Building 475D, Upton, NY 11973
[telephone: [phone]; Fax: [phone]].

2 pET System Vectors and Hosts

INTRODUCTION

pET Expression Vectors

The pET expression system1 is one of the most widely used systems for the
cloning and in vivo expression of recombinant proteins in E. coli. This is due
to the high selectivity of the pET system’s bacteriophage T7 RNA
polymerase for its cognate promoter sequences, the high level of activity of
the polymerase and the high translation efficiency mediated by the T7 gene
10 translation initiation signals. In the pET system, the protein coding
sequence of interest is cloned downstream of the T7 promoter and gene 10
leader sequences, and then transformed into E. coli strains. Protein
expression is achieved either by IPTG induction of a chromosomally
integrated cassette in which the T7 RNA polymerase is expressed from the
lacUV5 promoter, or by infection with the polymerase-expressing
bacteriophage lambda CE6.
expression of cloned target genes is extremely low in strains lacking a
source of T7 RNA polymerase. Upon induction the highly active
polymerase essentially out-competes transcription by the host RNA
polymerase. This phenomenon, together with high-efficiency translation,
achieves expression levels in which the target protein may constitute the
majority of the cellular protein—after only a few hours.

The pET expression vectors, derived from the pBR322 plasmid, are
engineered to take advantage of the features of the T7 bacteriophage gene
10 that promote high-level transcription and translation. The bacteriophage­encoded RNA polymerase is highly specific for the T7 promoter sequences,
which are rarely encountered in genomes other than T7 phage genome. First,
this ensures that the T7 promoter will not be recognized by host cell RNA
polymerase. Thus target genes are transcriptionally silent in the uninduced
state--a feature that is very important if the gene to be expressed is toxic to
the cell. Second, upon induction, the target gene is the only gene in the cell
that will be transcribed by the highly active polymerase.

In addition to the T7 promoter, all the vectors contain the gene 10 5´ leader,
which facilitates highly efficient translation. The protein coding sequence of
interest may be cloned directly after the gene 10 initiation codon using the
Nde I (pET-3,-11, a, b and c) or Nco I sites (pET-3d, and -11d).
Alternatively, the pET-3 and pET-11 vectors contain BamH I cloning sites
in all three reading frames relative to the gene 10 reading frame. Cloning the
gene of interest using the BamH I site results in a fusion protein containing
13 N-terminal amino acids from gene 10. The gene 10 transcription
terminator is also included downstream of the cloning sites to allow efficient
termination of transcription, preventing transcriptional read-through of
unwanted plasmid sequences and increasing the RNA polymerase density on
the sequence of interest--allowing high level accumulation of the specific
protein-coding RNA transcripts.

2

Due to the specificity of the T7 promoter, basal

pET System Vectors and Hosts 3

Despite the strong selectivity of the T7 promoter for its phage-encoded
polymerase, residual "leaky" expression of very toxic proteins from the
basic pET-3 constructs can be lethal to the cell. To circumvent this problem
and achieve more stringent control of expression, the lac operator has been
inserted between the T7 promoter and translation initiation sequences in the
pET-11 vectors, thereby allowing IPTG-mediated de-repression of the T7
promoter in addition to IPTG-induction of T7 polymerase from the lacUV5
promoter in the DE3 containing strains (see Bacterial Strains). In order to
provide adequate levels of lacI protein to shut off T7 polymerase expression
as well as T7 promoter transcription, the lacI

q

gene is included on the

pET-11 plasmids.

All of the vectors in the pET-3 and pET-11 series contain the β-lactamase
gene for ampicillin resistance and the pBR322 origin of replication. Other
features specific to the various vectors are listed in Table I below and in
Figures 1 and 2.

T

ABLE I

Features of the pET System Vectors

Vector Promoter ATG cloning

site

pET-3a T7 Nde I GGAa

pET-3b T7 Nde I GAT

pET-3c T7 Nde I ATC

pET-3d T7 Nco I ATC

pET-11a T7/lac O Nde I GGA

pET-11b T7/lac O Nde I GAT

pET-11c T7/lac O Nde I ATC

pET-11d T7/lac O Nco I ATC

a

Reading frame defined as the first full codon within the BamH I recognition sequence
(GGATCC) that is in the same frame as the gene 10 initiation codon.

Reading
frame

4 pET System Vectors and Hosts

pET-3 Vector Map

pET-3a–3d Cloning Site Regions

sequence shown (66–122)

Nde I

pET-3a

GAAGGAGATATACAT ATG GCT AGC ATG ACT GGT GGA CAG CAA ATG GGT CGC GGA TCC

RBS

M A S M T G G Q Q M G R G S

START

T7 gene 10 leader peptide

Nde I OR Nco I

RBS

P T7

gene 10 leader

BamH I

pET-3 vectors

4.6 kb

pBR322 ori

T T7

ampicillin

BamH I

BamH INde I

pET-3b

pET-3c

pET-3d

GAAGGAGATATACAT ATG GCT AGC ATG ACT GGT GGA CAG CAA ATG GGT CGG GAT CCG

RBS

GAAGGAGATATACAT ATG GCT AGC ATG ACT GGT GGA CAG CAA ATG GGT CGG ATC CGG

RBS

GAAGGAGATATACC ATG GCT AGC ATG ACT GGT GGA CAG CAA ATG GGT CGG ATC CGG C

RBS

M A S M T G G Q Q M G R D P

START

T7 gene 10 leader peptide

Nde I BamH I

M A S M T G G Q Q M G R I R

START

T7 gene 10 leader peptide

Nco I

M A S M T G G Q Q M G R I R

START

T7 gene 10 leader peptide

BamH I

Nucleotide Position

Feature

T7 promoter 1–19 1–19 1–19 1–19

ribosome binding site (RBS) 66–72 66–72 66–72 66–72

Nde I (pET-3a–c) or Nco I (pET-3d) cloning site 78–83 78–83 78–83 78–83

T7 gene 10 translated leader 81–113 81–113 81–113 80–112

BamH I cloning site 117–122 116–121 115–120 114–119

T7 terminator 191–237 190–236 189–235 188–234

ampicillin resistance (bla) ORF 840–1697 839–1696 838–1695 837–1694

pBR322 origin of replication 1848–2515 1847–2514 1846–2513 1845–2512

pET-3a pET-3b pET-3c pET-3d

FIGURE 1 The pET-3 vectors

pET System Vectors and Hosts 5

pET-11 Vector Map

RBS

P T7/lac O

Nde I OR Nco I

gene 10 leader

BamH I

T T7

pET-11a–11d Cloning Site Regions

sequence shown (74–130)

pET-11a

pET-11b

pET-11c

Nde I

GAAGGAGATATACAT ATG GCT AGC ATG ACT GGT GGA CAG CAA ATG GGT CGC GGA TCC

RBS

GAAGGAGATATACAT ATG GCT AGC ATG ACT GGT GGA CAG CAA ATG GGT CGG GAT CCG

RBS

Nde I BamH I

GAAGGAGATATACAT ATG GCT AGC ATG ACT GGT GGA CAG CAA ATG GGT CGG ATC CGG

RBS

Nhe I

M A S M T G G Q Q M G R G S

START

T7 gene 10 leader peptide

Nhe I

M A S M T G G Q Q M G R D P

START

T7 gene 10 leader peptide

Nhe I

M A S M T G G Q Q M G R I R

START

T7 gene 10 leader peptide

lacIq

pET-11 vectors

5.7 kb

pBR322 ori

BamH I

BamH INde I

ampicillin

BamH I

pET-11d

Nco I

GAAGGAGATATACC ATG GCT AGC ATG ACT GGT GGA CAG CAA ATG GGT CGG ATC CGG C

RBS

Nhe I

M A S M T G G Q Q M G R I R

START

T7 gene 10 leader peptide

Feature

pET-11a pET-11b pET-11c pET-11d

Nucleotide Position

T7 promoter with lac operator 1–43 1–43 1–43 1–43

ribosome binding site (RBS) 74–80 74–80 74–80 74–80

Nde I (pET-11a–c) or Nco I (pET-11d) cloning site 86–91 86–91 86–91 86–91

T7 gene 10 translated leader 89–121 89–121 89–121 88–120

BamH I cloning site 125–130 124–129 123–128 122–127

T7 terminator 199–245 198–244 197–243 196–242

ampicillin resistance (bla) ORF 657–1514 656–1513 655–1512 654–1511

pBR322 origin of replication 1665–2332 1664–2331 1663–2330 1662–2329

lacIq repressor ORF 4212–5291 4211–5290 4210–5289 4209–5288

FIGURE 2 The pET-11 vectors

6 pET System Vectors and Hosts

Bacterial Strains

The BL21-Gold expression strains provided with catalog #211621 and

-

#211623 are derivatives of the E. coli B strain BL21 (F- ompT r
The BL21 strain is generally good for protein expression due to its
deficiency in lon protease

3

as well as the ompT outer membrane protease

-

m

B

).

B

that can degrade proteins during purification. (For more information, see
Table II Features of the BL21-Derived Expression Strains.) BL21 strains are
rifampicin sensitive, allowing use of the drug to inhibit transcription of host
cell polymerase in instances where background synthesis is undesirable.

–

The strain BL21-Gold (DE3) [F

–

ompT hsdS(r

–

m

) dcm+ Tetr gal λ(DE3)

B

B

endA Hte] carries a lambda DE3 lysogen that has the phage 21 immunity
region, the lacI gene and the lacUV5-driven T7 RNA polymerase expression
cassette. Upon induction with IPTG, the lacUV5 promoter is de-repressed
allowing over-expression of T7 RNA polymerase and thus expression of the
T7-promoted target gene from the pET expression plasmid.

In order to further reduce basal activity of T7 RNA polymerase in the
uninduced state, the strain BL21-Gold(DE3) [F

r

Tet

gal λ(DE3) endA Hte (pLysS Camr)] carries a derivative of the plasmid

–

ompT hsdS(r

B

–

m

–

) dcm+

B

pACYC184, which expresses the T7 lysozyme gene at low levels. T7
lysozyme binds to T7 RNA polymerase and inhibits transcription by this
enzyme. Upon IPTG induction, overproduction of the T7 RNA polymerase
renders low level inhibition by T7 lysozyme virtually ineffective. In addition
to inactivation of T7 RNA polymerase transcription, T7 lysozyme has a
second function involving specific cleavage of the peptidoglycan layer of
the E. coli outer wall. The inability of T7 lysozyme to pass through the
bacterial inner membrane restricts the protein to the cytoplasm, allowing
E. coli to tolerate expression of the lysozyme protein. This second function
of lysozyme does, however, confer the advantage of allowing cell lysis
under mild conditions. Cells expressing T7 lysozyme are subject to lysis
under conditions that would normally only disrupt the inner membrane (e.g.,
freeze–thaw cycles or the addition of chloroform or a mild detergent such as

0.1% Triton

®

X-100) due to the action of the protein on the outer wall when

the inner membrane is disrupted.

The BL21-Gold expression strains incorporate major improvements over the
original BL21 strain. The BL21-Gold strains feature the Hte phenotype
present in our highest efficiency strain, XL10-Gold ultracompetent cells.

4

The presence of the Hte phenotype increases the transformation efficiency
of the BL21-Gold cells to >1 × 10

8

cfu/μg of pUC18 DNA. In addition, the

gene that encodes endonuclease I (endA), which rapidly degrades plasmid
DNA isolated by most miniprep procedures, is inactivated. These two
improvements allow direct cloning of many protein expression constructs.

pET System Vectors and Hosts 7

In cases where the protein of interest is extremely toxic to E. coli or where
clones obtained from direct transformation of BL21-Gold strains
consistently show sequence anomalies, perform the initial cloning steps in
cells in which the T7 promoter is inactive and that are RecA-(e.g. XL1-Blue
competent cells). Strains carrying the lacI

q

gene may be useful for further

inhibiting basal expression from the pET-11 vectors during subcloning.

T

ABLE II

5

Features of the BL21-Derived Expression Strains

Expression Strain Induction Advantages Disadvantages

BL21-Gold (DE3)
competent cells

BL21-Gold (DE3)pLysS
competent cells

BL21 competent cells Infection with lambda

IPTG induction of T7
polymerase from lacUV5
promoter

IPTG induction of T7 Ease of induction,

bacteriophage CE6

High-level expression;

Ease of induction, direct
cloning in expression
strain

reduced uninduced
expression, direct cloning
in expression strain

Tightest control of
uninduced expression

Leaky expression of T7
polymerase can lead to
uninduced expression of
potentially toxic proteins.

Slight inhibition of induced
expression when compared
with BL21-Gold (DE3).

Induction not as efficient as
DE3 derivatives. Induction
(infection) process is more
cumbersome.

Bacteriophage CE6

In cases where target genes are too toxic to allow plasmids to be established
in DE3 lysogens, T7 RNA polymerase can be delivered to the cell by
infection with the bacteriophage CE6. By this method, no T7 RNA
polymerase is present in the cell until the desired time of induction. CE6
expresses T7 RNA polymerase from the lambda pL and pI promoters, and
carries the Sam7 lysis mutations. This bacteriophage will allow effective
expression of target genes in BL21 cells, and presumably other
nonrestricting hosts which absorb lambda. The phage can be propagated in
the host strain LE392 [F-e14- (mcrA) hsdR514 (r
which suppresses the Sam7 mutation and therefore allows lysis of
infected cells.

+

-

) supE44 supF58],

m

k

k

6

8 pET System Vectors and Hosts

CLONING PROTOCOL

Preparing the Vectors

♦ Perform a complete DNA digestion with the appropriate enzymes. (Perform

Nde I–BamH I or Nco I–BamH I double digests to clone the gene of interest
at the initiation codon. Perform a BamH I single digest to produce a fusion of
the protein of interest to the gene 10 translated leader.) If the inserts to be
cloned into these vectors contain one or more internal Nde I, Nco I or
BamH I sites, PCR primers may be engineered to include restriction sites
with overhangs compatible with the overhangs produced by the chosen
enzymes.

♦ Dephosphorylate the digested pET vector with calf intestinal alkaline

phosphatase (CIAP) prior to ligating to the insert DNA. If more than one
restriction enzyme is used, the background can be reduced further by
electrophoresing the digested vector DNA on an agarose gel and gel
purifying the desired vector band, leaving behind the small fragment excised
from between the two restriction enzyme sites.

♦ After gel purification, resuspend the vector DNA in a volume of TE buffer

(see Preparation of Media and Reagents) that will allow the concentration of
the vector DNA to be the same as the concentration of the insert DNA

(~0.1μg/μl).

pET System Vectors and Hosts 9

Ligating the Insert

For ligation, the ideal insert-to-vector ratio of DNA is variable; however, a
reasonable starting point is 2:1 (insert-to-vector molar ratio), measured in
available picomole ends. This is calculated as follows:

1. Prepare three control and two experimental 10-μl ligation reactions by

a

low number of transformant colonies if the digestion and CIAP treatment are effective.

b

vector remains. Expect an absence of transformant colonies if the digestion is complete.

c

absence of transformant colonies if the insert is pure.

d

the transformant colonies to represent recombinants.

e

6

10 2

DNAof gramends/micro Picomole

=

×

600 pairs base ofnumber

×

adding the following components to separate sterile 1.5-ml
microcentrifuge tubes:

Note For blunt-end ligation, reduce the rATP to 0.5 mM and

incubate the reactions overnight at 12–14°C.

Control Experimental

Ligation reaction components 1a 2

Prepared vector (0.1 μg/μl) 1.0 μl 1.0 μl 0.0 μl 1.0 μl 1.0 μl

Prepared insert (0.1 μg/μl) 0.0 μl 0.0 μl 1.0 μl X μl X μl

rATP [10 mM (pH 7.0)] 1.0 μl 1.0 μl 1.0 μl 1.0 μl 1.0 μl

Ligase buffer (10×)e 1.0 μl 1.0 μl 1.0 μl 1.0 μl 1.0 μl

T4 DNA ligase (4 U/μl) 0.5 μl 0.0 μl 0.5 μl 0.5 μl 0.5 μl

Double-distilled (ddH2O) to 10 μl 6.5 μl 7.0 μl 6.5 μl X μl X μl

This control tests for the effectiveness of the digestion and the CIAP treatment. Expect a

This control indicates whether the vector is cleaved completely or whether residual uncut

This control verifies that the insert is not contaminated with the original vector. Expect an

These experimental ligation reactions vary the insert-to-vector ratio. Expect a majority of

See Preparation of Media and Reagents.

b

3

c

4

d

5

d

2. Incubate the reactions for 2 hours at room temperature (22°C) or
overnight at 4°C.

3. Transform 1–2 μl of the ligation mix into the appropriate competent
bacteria. Plate on selective media.

Note For most applications, the ligation reaction may be

transformed directly into BL21-Gold(DE3) or
BL21-Gold(DE3)pLysS competent cells (provided with
catalog #211621 and #211623). See Transformation of the

BL21-Gold Expression Strains for a transformation protocol.

4. Verify the presence and orientation of the insert by PCR, restriction
analysis or other appropriate methods.

10 pET System Vectors and Hosts

TRANSFORMATION PROTOCOL

Transformation of the BL21-Gold Expression Strains

1. Thaw the competent cells on ice.

Note Store the competent cells on ice at all times while aliquoting.

It is essential that the Falcon 2059 polypropylene tubes are
placed on ice before the competent cells are thawed and that

μ

l of competent cells are aliquoted directly into each

100
prechilled polypropylene tube. Do not pass the frozen
competent cells through more than one freeze–thaw cycle.

2. Gently mix the competent cells. Aliquot 100 μl of the competent cells
into the appropriate number of prechilled 15-ml Falcon 2059
polypropylene tubes.

3. Add 1–50 ng of DNA to each transformation reaction and swirl gently.
For the control transformation reaction, add 1 μl of the pUC18 control

plasmid (provided with Catalog #211621 and #211623) to a separate
100-μl aliquot of the competent cells and swirl gently.

4. Incubate the reactions on ice for 30 minutes.

5. Preheat SOC medium

§

in a 42°C water bath for use in step 8.

6. Heat-pulse each transformation reaction in a 42°C water bath for
20 seconds. The duration of the heat pulse is critical for optimal

transformation efficiencies.

7. Incubate the reactions on ice for 2 minutes.

8. Add 0.9 ml of 42°C SOC medium to each transformation reaction and
incubate the reactions at 37°C for 1 hour with shaking at 225–250 rpm.

9. Concentrate the cells from the experimental transformation by
centrifugation (200 × g for 3–5 minutes) and plate the entire
transformation reaction (using a sterile spreader)
LB–ampicillin agar plate.

§

ll

onto a single

To plate the cells transformed with the pUC18 control plasmid, first
place a 195-μl pool of SOC medium on an LB–ampicillin agar plate.

§

Add 5 μl of the control transformation reaction to the pool of SOC
medium. Use a sterile spreader to spread the mixture.

10. Incubate the plates overnight at 37°C.

§

See Preparation of Media and Reagents.

ll

When spreading bacteria onto the plate, tilt and tap the spreader to remove the last drop of

cells. If plating <100 μl of the transformation reaction, plate the cells in a 200-μl pool of
SOC medium. If plating ≥100 μl, the cells can be spread directly onto the plates.

pET System Vectors and Hosts 11

Transformation Summary for the pUC18 Control Plasmid

Host strain

BL21-Gold(DE3)
competent cells

BL21-Gold(DE3)pLysS
competent cells

a

The efficiencies quoted for the BL21-Gold(DE3) and the BL21-Gold(DE3)pLysS host

strain competent cells (provided with Catalog #211621 and #211623) are
guaranteed only if the user follows the storage and transformation protocols outlined in
this manual. Efficiencies of competent cells prepared by the user cannot be guaranteed.

EXPRESSION PROTOCOLS

Induction of Target Protein Using IPTG

The following induction protocol is a general guide for expression of genes
under the control of IPTG-inducible promoters on an analytical scale (1 ml
of induced culture). Most commonly, this protocol is used to analyze protein
expression of individual transformants when using BL21-Gold(DE3) host
strains in combination with plasmids containing T7 promoter constructs
(e.g. pET vectors). Expression cassettes under the control of the trp/lac
hybrid promoter, tac, can be also induced using this protocol. In the case of
tac promoter constructs, non-DE3 lysogen strains can be employed as hosts.

Plating
quantity

5 μl >50 ≥1 × 108

5 μl >50 ≥1 × 108

Expected
number of
colonies

Efficiency
(cfu/μg of
pUC18 DNA)

Note The transformation procedure described above will produce

varying numbers of colonies depending on the efficiency of
transformation obtained using the expression plasmid. It is
prudent to test more than one colony as colony-to-colony
variations in protein expression are possible.

1. Inoculate 1-ml aliquots of LB broth

§

containing 100 μg/ml of

carbenicillin or ampicillin with single colonies from the transformation.
Shake at 220–250 rpm at 37°C overnight.

Note If the transformed cells contain a pACYC-based plasmid

(e.g., the BL21-Gold(DE3)pLysS strain or any
BL21-CodonPlus strain), the overnight culture must include

μ

chloramphenicol at a final concentration of 50

g/ml in
addition to the carbenicillin/ampicillin required to maintain
the pET plasmid.

2. The next morning, pipet 50 μl of each culture into fresh 1-ml aliquots

of LB broth containing no selection antibiotics. Incubate these cultures
with shaking at 220–250 rpm at 37°C for 2 hours.

§

See Preparation of Media and Reagents.

12 pET System Vectors and Hosts

3. Pipet 100 μl of each of the cultures into clean microcentrifuge tubes

and place the tubes on ice until needed for gel analysis. These will
serve as the non-induced control samples.

4. To the rest of the culture in each tube add IPTG to a final concentration
of 1 mM. Incubate with shaking at 220–250 rpm at 37°C for 2 hours.

Note These values for IPTG concentration and induction time are

starting values only and may require optimization depending
on the gene expressed.

5. After the end of the induction period, place the cultures on ice.

6. Pipet 20 μl of each of the induced cultures into clean microcentrifuge

§

tubes. Add 20 μl of 2× SDS gel sample buffer

to each.

7. Mix the tubes containing the non-induced samples to resuspend the
cells and pipet 20 μl from each tube into clean microcentrifuge tubes.
Add 20 μl of 2× SDS gel sample buffer to each.

8. Heat all tubes to 95°C for 5 minutes and analyze the samples by
Coomassie

®

Brilliant Blue staining of an SDS-PAGE gel, placing

associated non-induced/induced samples in adjacent lanes.

Induction of Target Protein by Infection with Lambda CE6

Expression of genes under the control of the T7 promoter (e.g. genes in pET
vectors) can be achieved in non-DE3 lysogen host strains (e.g. BL21) if the
strain harboring the expression plasmid is subsequently infected with
lambda CE6. Lambda CE6 expresses T7 polymerase, which in turn drives
the transcription of the gene downstream of the T7 promoter. The following
protocols describe the growth and maintenance of lambda CE6 and the use
of lambda CE6 for infecting host strains. We offer the Lambda CE6
Induction Kit (Stratagene Catalog# 235200) for use in protein expression
protocols which incorporate CE6 infection.

§

See Preparation of Media and Reagents.

pET System Vectors and Hosts 13

Growth and Maintenance of High-Titer Bacteriophage Lambda
CE6 Stocks

1. Inoculate 5 ml of modified* NZY broth§ with a single colony of LE392
host cells. Shake overnight at 37°C at 220–250 rpm.

2. Centrifuge the overnight culture for 15 minutes at 1700–2000 × g at
4°C. Resuspend the cells in 10 mM MgSO

to a final OD

4

600

of 0.5.

3. Combine 250 μl of cells (at OD

4. Add 3 ml of melted NZY top agar

5. Flood each plate with 5 ml of SM solution

6. Remove the SM solution (which contains the lambda CE6) from each

7. Centrifuge the SM solution at 1700–2000 × g for 15 minutes at 4°C.

8. Remove the supernatant and determine the titer of the solution.

9. Store the lambda CE6 stock at 4°C.

Phage Amplification

If the titer drops over time, or if more phage are needed, grow up LE392
cells in 10 ml of medium and add bacteriophage lambda CE6 at a
multiplicity of infection of 1:1000 (CE6-to-cell ratio). Continue growing the
culture at 37°C for 5–6 hours and spin down the cellular debris. Titer of the
supernatant should be
phage amplification, see reference 7.

= 0.5) with 1 × 106 pfu of CE6 in

600

Falcon

®

2059 polypropylene tubes in triplicate. Incubate at 37°C for

15 minutes.

§

(equilibrated to 48°C) to each cell

suspension and plate on warm agarose plates.

§

overnight at 37°C.

§

and rock the plates for

2 hours at room temperature.

plate and pool the volumes in a 50-ml conical tube.

≥5.0 × 10

9

pfu/ml. For general information regarding

Incubate the plates

Induction of Target Protein by Infection with Lambda CE6

Note This protocol is designed for inductions in 50-ml culture volumes.

If inductions of larger volumes of culture are desired, it will be
necessary to increase the volume of the overnight culture in step 1.
The increased volume of overnight culture is necessary to achieve

the required cell density (A
the following day.

* NZY broth to be used for lambda infection protocols should be supplemented with maltose

at a final concentration of 0.2%. Add 1 ml of 20% maltose solution (filter-sterilized) per
100 ml of NZY broth to achieve the correct final concentration of maltose.

§

See Preparation of Media and Reagents.

14 pET System Vectors and Hosts

≤ 1) in the larger volume of broth

600

1. Inoculate 5 ml of modified* NZY broth containing 100 μg/ml of
carbenicillin or ampicillin with a single colony of BL21 cells (not a
DE3 lysogen) harboring the expression plasmid. Shake overnight at
37°C at 200–250 rpm.

Note If the host cells contain a pACYC-based plasmid (e.g., any

BL21-CodonPlus strain), the overnight culture must include
chloramphenicol at a final concentration of 50

μ

g/ml in
addition to the carbenicillin/ampicillin required to maintain
the pET plasmid.

2. In the morning, centrifuge 1.0 ml of the overnight culture, resuspend
the cells in 1.0 ml of fresh modified* NZY broth, and pipet the
resuspended cells into a flask containing 50 ml of fresh modified* NZY
broth (no selection antibiotics).

3. Record the A
> 0.1, use more fresh modified* NZY broth to dilute the culture to A

≤ 0.1. If the A

of the diluted culture. It should be ≤ 0.1. If the A

600

is < 0.1, the time required to reach an A

600

of 0.3 (in

600

600

is

600

step 4) will be extended.

4. Grow the culture to an A

of 0.3 and add glucose to a final

600

concentration of 4 mg/ml (e.g. 1.0 ml of a 20% glucose solution to the
50-ml culture).

5. Grow the culture to an A
concentration of 10 mM (e.g. 500 μl of a 1.0 M solution of MgSO

of 0.6–1.0 and add MgSO4 to a final

600

4

to

the 50-ml culture).

6. Remove a portion of the culture to serve as the uninduced control and
infect the rest with bacteriophage lambda CE6 at a multiplicity of
infection (MOI) of 5–10 particles per cell. (To optimize induction,
cultures may be split into 3 or 4 aliquots and infected with varying
dilutions of bacteriophage lambda CE6. The subsequent induction can
be monitored by SDS-PAGE or by a functional assay, if available.)

7. Grow the culture for 2–3 hours.

8. Remove 5–20 μl of the culture for determination by SDS-PAGE, and
harvest the remaining culture by centrifugation. Store the pellets at
–70°C.

Note If induction will be monitored using Coomassie stain, silver

stain, or another nonspecific protein stain, include a control
of CE6-infected BL21 cells harboring the plasmid without a
cloned insert.

* NZY broth to be used for lambda infection protocols should be supplemented with maltose

at a final concentration of 0.2%. Add 1 ml of 20% maltose solution (filter-sterilized) per
100 ml of NZY broth to achieve the correct final concentration of maltose.

pET System Vectors and Hosts 15

TROUBLESHOOTING

Observation Suggestions

Poor induction Poor induction can occur for a number of reasons, including loss of plasmid

due to instability resulting from expression of toxic proteins, unstable mRNA or
poorly translated RNA that is high in secondary structure, and high abundance
of codons which are rare in E. coli. BL21-CodonPlus Competent Cells are
engineered to contain extra copies of genes that encode the tRNAs that most
frequently limit translation of heterologous proteins in E. coli.

Plasmid instability

Problems associated with
induction time

Inclusion bodies In some cases protein may form insoluble inclusion bodies at 37°C. In many

Prior to induction of cultures, determine the fraction of cells containing inducible
plasmid by plating a 105–fold dilution of cells on plates that have a) IPTG plus
ampicillin, b) IPTG alone, and cells at a dilution of 2 × 10
c) ampicillin or d) nothing

Reduced colony formation should occur on ampicillin-containing plates relative
to the LB plates. Successful induction of target gene on IPTG-containing plates
should result in virtually no increase in bacterial growth; therefore, <2% of the
cells should form colonies on plates that contain IPTG alone and 0.01% on
plates that contain IPTG plus antibiotic. Conversely, cells that contain mutant
plasmids that are poorly induced should show higher colony formation on the
IPTG plates

More tightly controlled induction may be desirable. Available methods include
using a pET-11 vector (instead of a pET-3 vector) containing a lac operator;
using BL21-Gold(DE3)pLysS (instead of BL21-Gold(DE3)); and, in extreme
cases, performing induction by infection with the bacteriophage CE6. Also see
Table II, Features of the BL21-Derived Expression Strains

In certain cases accumulation of target protein may kill cells at saturation while
allowing normal growth in logarithmically growing cultures, while in other cases
target protein may continue to accumulate in cells well beyond the
recommended 3-hour induction period. To determine the optimal time of
induction, a time course may be carried out during which a small portion of the
culture is analyzed by SDS-PAGE at various times following induction

cases, this protein may be soluble and active if the induction is carried out at
30°C. Inclusion body formation may be used as a purification step by simply
spinning out the insoluble material from crude lysates and redissolving the
protein in urea or guanidinium-HCl

6

–fold which have

16 pET System Vectors and Hosts

PREPARATION OF MEDIA AND REAGENTS

LB Broth (per Liter)

10 g of NaCl
10 g of tryptone
5 g of yeast extract
Adjust to pH 7.0 with 5 N NaOH
Add deionized H2O to a final volume of

1 liter

Autoclave

LB–Ampicillin Broth (per Liter)

1 liter of LB broth, autoclaved
Cool to 55°C
Add 10 ml of 10-mg/ml filter-sterilized

ampicillin

LB–Ampicillin–Methicillin Agar (per
Liter)

(Use for reduced satellite colony formation)
1 liter of LB agar
Autoclave
Cool to 55°C
Add 20 mg of filter-sterilized ampicillin
Add 80 mg of filter-sterilized methicillin
Pour into petri dishes (~25 ml/100-mm

plate)

LB Agar (per Liter)

10 g of NaCl
10 g of tryptone
5 g of yeast extract
20 g of agar
Adjust pH to 7.0 with 5 N NaOH
Add deionized H2O to a final volume of

1 liter
Autoclave
Pour into petri dishes (~25 ml/100-mm

plate)

LB–Ampicillin Agar (per Liter)

1 liter of LB agar, autoclaved
Cool to 55°C
Add 10 ml of 10-mg/ml filter-sterilized

ampicillin
Pour into petri dishes

(~25 ml/100-mm plate)

NZY broth, (per Liter)

5 g of NaCl
2 g of MgSO
5 g of yeast extract
10 g of NZ amine (casein hydrolysate)
Adjust the pH to 7.5 with NaOH

. 7H2O

4

TE Buffer

10 mM Tris-HCl (pH 7.5)
1 mM EDTA

SOB Medium (per Liter)

20.0 g of tryptone

5.0 g of yeast extract

0.5 g of NaCl
Autoclave
Add 10 ml of 1 M MgCl
1 M MgSO

to use

Filter sterilize

pET System Vectors and Hosts 17

/liter of SOB medium prior

4

and 10 ml of

2

SOC Medium (per 100 ml)

SOB medium
Add 1 ml of a 2 M filter-sterilized glucose

solution or 2 ml of 20% (w/v) glucose

prior to use
Filter sterilize

10× Ligase Buffer

500 mM Tris-HCl (pH 7.5)
70 mM MgCl
10 mM dithiothreitol (DTT)

Note rATP is added separately in the ligation

reaction.

2

Agarose Plates (per Liter)

Melt 20 g of agarose in 500 ml of deionized

O

H

2

Add the following:
5 g of NaCl
5 g of yeast extract
10 g of tryptone
Add deionized H

1 liter
Autoclave
Pour into petri dishes (~25 ml/100-mm plate)

O to a final volume of

2

NZY Top Agar (per Liter)

Prepare 1 liter of NZY broth
Add 0.7% (w/v) agarose
Autoclave

SM Solution

5 g of NaCl
2 g of MgSO
50 ml of 1 M Tris-HCl (pH 7.5)
5 ml 2% gelatin
Add deionized H

1 liter
Adjust the pH to 7.5
Autoclave

. 7H2O

4

O to a final volume of

2

2× SDS gel sample buffer

100 mM Tris-HCl (pH 6.5)
4% SDS (electrophoresis grade)

0.2% bromophenol blue
20% glycerol

Note Add dithiothreitol to a final

concentration in the 2× buffer of
200 mM prior to use. This sample

buffer is useful for denaturing,
discontinuous acrylamide gel
systems only.

18 pET System Vectors and Hosts

REFERENCES

ENDNOTES

1. Studier, F. W., Rosenberg, A. H., Dunn, J. J. and Dubendorff, J. W. (1990) Methods
Enzymol 185:60–89.

2. Studier, F. W. and Moffatt, B. A. (1986) J Mol Biol 189(1):113–30.

3. Phillips, T. A., VanBogelen, R. A. and Neidhardt, F. C. (1984) J Bacteriol 159(1):283–

7.

4. Jerpseth, B., Callahan, M. and Greener, A. (1997) Strategies 10(2):37–38.

5. Weiner, M. P., Anderson, C., Jerpseth, B., Wells, S., Johnson-Browne, B. et al. (1994)
Strategies 7(2):41–43.

6. Borck, K., Beggs, J. D., Brammar, W. J., Hopkins, A. S. and Murray, N. E. (1976) Mol
Gen Genet 146(2):199–207.

7. Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory
Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Triton® is a registered trademark of Rohm and Haas Co.
Coomassie
Falcon

®

is a registered trademark of Imperial Chemical Industries.

®

is a registered trademark of Becton-Dickinson and Co.

MSDS INFORMATION

The Material Safety Data Sheet (MSDS) information for Stratagene products is provided on the web at
http://www.stratagene.com/MSDS/. Simply enter the catalog number to retrieve any associated MSDS’s
in a print-ready format. MSDS documents are not included with product shipments.

pET System Vectors and Hosts 19

20

21

pET System Vectors and Hosts

Catalog #211521, #211523, #211621, and #211623

QUICK-REFERENCE PROTOCOL

♦ Prepare DNA insert containing the target gene

♦ Prepare pET vector by digestion with the appropriate restriction enzyme(s), CIAP treatment and

agarose gel purification

♦ Ligate the prepared vector and insert DNA

♦ Transform cloning host competent cells (e.g. Stratagene XL1-Blue cells) and verify construct

Expression in E. coli

♦ Transform BL21 expression host

competent cells

♦ Induce protein expression

by IPTG addition or CE6 infection

Expression in vitro

♦ Add recombinant pET plasmid DNA

or PCR-amplified DNA to coupled
transcription/translation reaction

♦ Purify target protein

♦ Harvest cells; analyze cell pellets for

target protein production

♦ Purify target protein

22