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ACEMBL Expression System Series
MultiColi
Multi-Protein Expression for E.coli
User Manual
Vers. 2.0
June 2011
This manual is based on the original ACEMBL manual (November 2009) written by
Yan Nie, Christoph Bieniossek and Imre Berger but has been revised, updated and,
wherever necessary, modified and expanded to meet customer demands.
ACEMBL was developed at the European Molecular Biology Laboratory, EMBL
Grenoble Outstation, Grenoble, France.
2
Table of Contents
A. The ACEMBL System Kit: Contents and Storage
3
B. Introduction
4
C. Synopsis
5
C. The ACEMBL System
6
C.1. ACEMBL vectors
C.2. The multiple integration element (MIE)
C.3. Tags, promoters, terminators
C.4. Generating Plasmid Constructs for Complex Expression
C.5. Complex Expression
D. Procedures
D.1. Cloning into ACEMBL vectors
D.1.1. Single gene insertion into the MIE by SLIC
D.1.2. Polycistron assembly in MIE by SLIC
D.1.3. Gene insertion by restriction/ligation
D.1.4. Multiplication by using the HE and BstXI sites
D.2. Cre-LoxP reaction of Acceptors and Donors
D.2.1. Cre-LoxP fusion of Acceptors and Donors
D.2.2. Deconstruction of fusion vectors by Cre
D.3. Co-expression by Co-transformation
E. ACEMBL multi-gene combination: Examples
E.1. SLIC cloning into ACEMBL vectors: human TFIIF
E.2. The Homing endonuclease/BstXI module: yeast RES complex
F. Appendix
F.1. DNA sequence of MIE
F.2. DNA sequences of ACEMBL vectors
F.2.1. pACE1
F.2.2. pACE2
F.2.3. pDC
F.2.4. pDK
F.2.5. pDS
F.3. ACEMBL plasmid maps
F.4. Analytical restriction of ACEMBL vectors
G. References
ACEMBL MultiColi
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A. The ACEMBL System Kit: Contents and Storage
Reagents supplied in ACEMBL system kit:
Acceptor vectors: pACE1, pACE2 – approx. 2 µg plasmid DNA per vial
Donor vectors: pDC, pDK, pDS – approx. 2 µg plasmid DNA per vial
keep at 4°C for short-term storage and in a freezer at -20°C or lower for medium- and
long-term storage (take care to avoid repeated freeze-thaw cycles, e.g. by aliquotting
DNA prior to freezing)
E. coli transformed with Acceptor and Donor vectors, provided as agar
stabs; for plating bacteria as a starting point for plasmid preparations
keep agar stabs at 4°C or room temperature
pirLC, pirHC cells†
†
E. coli strains expressing the pir gene product for propagation of donor vectors (any
+
other strain with pir background can be used as well). LC: low copy number
propagation, HC: high copy number propagation of plasmids with R6K origin.
keep agar stabs at 4°C or room temperature
Additionally required reagents:
Antibiotics: ampicillin, chloramphenicol, kanamycin, spectinomycin, tetracycline
Enzymes: Cre recombinase
T4 DNA polymerase (for recombination insertion of genes)
Phusion polymerase or any other proof-reading DNA polymerase (for
PCR amplification of DNA)
Restriction enzymes and T4 DNA ligase (for restriction-ligation cloning)
Standard laboratory E.coli strain for cloning (TOP10, HB101, DH5)
Expression strain(s) of choice, e.g. BL21(DE3), Rosetta, AD494, Origami (DE3), etc.
Choice of strain will depend on your target protein and its underlying DNA sequence.
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B. Introduction
Protein complexes are the heart and soul of many cellular processes 1 . Some
researchers go as far as describing the cell as “a collection of protein machines”2.
Whether you think of replication, transcription 3 , translation 4 , DNA repair, the
processing, import, trafficking as well as export of proteins or other biomolecules, or
the maintenance of the structural stability and integrity of any cell, multi-subunit
protein assemblies play an important role in all these biological phenomena.
In addition, other processes, e.g. entry of viruses into human cells, also critically
hinge on multiple proteins or protein complexes5. Moreover, various prokaryotic
microorganisms, with E.coli being the prototypical workhorse, are harnessed to
express heterologous proteins and protein complexes but also to cost-efficiently
produce known or novel compounds by means of metabolic engineering6.
Scientists wishing to study these processes in functional and structural detail, often
require significant amounts of the protein complexes under investigation. While
obtaining bulk protein usually is not a problem for protein complexes that are
abundant in a steady-state cell, this becomes more difficult for complexes that are
transient in nature, appear only periodically in cells or simply occur only in low
abundance. In such cases, systems come in handy that allow homo- or heterologous
expression of these complexes in large amounts.
While various methods and systems have been developed to address this problem,
most of them are of little use for intense research efforts directed at generating and
investigating scores of protein complexes in parallel, i.e. in an automated fashion.
Such a system should be robust and easy-to-install in terms of manipulation steps /
protocols and/or components used in the process7. The ACEMBL system exactly
addresses these needs.
1
Robinson et al., Nature 450, 973 (2007); Charbonnier S et al., Biotechnol Annu Rev 14, 1 (2008)
Alberts, Cell 92, 291 (1998).
3
Van Hijum et al., Microbiol Mol Biol Rev 73, 481 (2009).
4 Estrozi et al., Nat Struct Mol Biol 18, 88
5
Bhattacharya, Nature 459, 24 (2009).
6
Chemler and Koffas, Curr Opin Biotech 19, 597 (2008); Chou, Appl Microbiol Biotech 76, 521 (2007);
Lee et al., Curr Opin Biotech 19, 553 (2008).
7
Nie et al., Curr Genomics 10: 558-72 (2009).
2
ACEMBL MultiColi
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C. Synopsis
ACEMBL MultiColi is a 3rd generation multi-gene expression system for complex
production in E. coli, created at the European Molecular Biology Laboratory EMBL,
at Grenoble. ACEMBL can be applied both manually and also in an automated set-up
by using a liquid handling workstation. ACEMBL applies tandem recombination steps
for rapidly assembling many genes into multi-gene expression cassettes. These can
be single or polycistronic expression modules, or a combination of these elements.
ACEMBL also offers the option to employ conventional approaches involving
restriction enzymes and ligases if desired, which may be the methods of choice in
laboratories not familiar with recombination approaches.
The following strategies for multi-gene assembly and expression are
provided for in the ACEMBL system and detailed in Sections C and D:
(1) Single gene insertions into vectors (recombination or restriction/ligation)
(2) Multi-gene
assembly
into
a
polycistron
(recombination
or
restriction/ligation)
(3) Multi-gene assembly using homing endonucleases
(4) Multi-gene plasmid fusion by Cre-LoxP reaction
(5) Multi-gene expression by cotransformation
These strategies can be used individually or in conjunction, depending on the project
and user.
In Section D, step-by-step protocols are provided for each of the methods for
multi-gene cassette assembly that can be applied in the ACEMBL MultiColi system.
Each procedure is illustrated by corresponding complex expression experiments in
Section D of this Supplement.
DNA sequences of ACEMBL vectors are provided in the Appendix and can be
copied from there for further use.
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C. The ACEMBL System
C.1. ACEMBL vectors
At the core of the technology are five small de novo designed vectors which are
called “Acceptor” and “Donor” vectors (see Illustration 1). Acceptor vectors (pACE1,
pACE2) contain origins of replication derived from ColE1 (low to medium copy) and
resistance markers (ampicillin or tetracycline). Donor vectors contain conditional
origins of replication (derived from phage R6K), which make their propagation
dependent on hosts expressing the pir gene. Donor vectors contain resistance
markers kanamycin, chloramphenicol, or spectinomycin. Up to three Donor vectors
can be used in conjunction with one Acceptor vector.
Illustration 1: ACEMBL system for multi-protein complex production
All Donor and Acceptor vectors contain a loxP imperfect inverted repeat and
in addition, a multiple integration element (MIE). This MIE consists of an expression
cassette with a promoter of choice (prokaryotic, mammalian, insect cell specific or a
combination thereof, depending on the ACEMBL system) and a corresponding
terminator (if required, e.g. the lac promoter does not require a matching
terminator). These flank a DNA segment that contains a number of restriction sites
which can be used for conventional cloning approaches or also for generating
double-strand breaks for the integration of expression elements of choice (further
promoters, ribosomal binding sites, terminators and genes). The MIE is completed
ACEMBL MultiColi
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by a homing endonuclease site and a specifically designed restriction enzyme site
(BstXI) flanking the promoter and the terminator (see C.2.) Vector DNA sequences
are provided in the Appendix. Maps of all vectors are shown at the end of this
manual.
C.2. The multiple integration element (MIE)
Illustration 2: The multiple integration element, schematic representation.
The MIE was derived from a polylinker8 and allows several approaches for multigene assembly (Section D). Multiple genes can be inserted into the MIE of any one
of the vectors by a variety of methods, for example BD-In-Fusion recombination9 or
SLIC (sequence and ligation independent cloning)10. For this, the vector needs to be
linearized, which can also be carried out efficiently by PCR reaction with appropriate
primers, since the vectors are all small (2 to 3 kb). Use of ultrahigh-fidelity
polymerases such as Phusion11 is recommended. Alternatively, if more conventional
approaches are preferred, i.e. in a regular wet lab setting without robotics, the
vectors can also be linearized by restriction digestion, and a gene of interest can be
integrated by restriction / ligation (Section D). The DNA sequence of the MIE is
shown in the Appendix.
8
Tan et al. Protein Expr. Purif. 40, 385 (2005)
ClonTech TaKaRa Bio Europe, www.clontech.com
10
Li and Elledge, Nat. Methods 4, 251 (2007)
11
Finnzymes/New England BioLabs, www.neb.com
9
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C.3. Tags, promoters, terminators
Current vectors of the ACEMBL system for Escherichia coli contain the default
promoters T7 and Lac, as well as the T7 terminator element (Illustr.1, 10). The T7
system is currently most commonly used; it requires bacterial strains which contain
a T7 polymerase gene in the E. coli genome. The Lac promoter is a strong
endogenous promoter which can be utilized in most strains. All ACEMBL vectors
contain the lac operator element for repression of heterologous expression.
Evidently, all promoters and terminators present in ACEMBL Donor and
Acceptor vectors, and in fact the entire multiple integration element (MIE) can be
exchanged with a favored expression cassette by using restriction/ligation cloning
with appropriate enzymes (for example ClaI/PmeI, Illustration 2) or insertion into
linearized ACEMBL vectors where the MIE was removed by sequence and ligation
independent approaches such as SLIC (sequence and ligation independent cloning).
In an experimental variation the T7 promoter in pDC was substituted with a trc
promoter (pDCtrc) and the T7 promoter in pACE with an arabinose promoter
(pACEara). The resulting vectors were used successfully in co-expression experiments
by inducing with arabinose and IPTG.
Currently, the ACEMBL system vectors do not contain DNA sequences
encoding for affinity tags that enable purification or solubilization of the protein(s)
of interest. We typically use C- or N-terminal oligohistidine tags, with or without
protease sites for tag removal. We introduce these by means of the respective PCR
primers used for amplification of the genes of interest prior to SLIC mediated
insertion. We recommend outfitting Donors or Acceptors of choice by the array of
custom tags that are favored in individual user laboratories prior to inserting
recombinant genes of interest. This is best done by a design which will, after tag
insertion, still be compatible with the recombination based principles of ACEMBL
system usage.
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C.4. Generating Plasmid Constructs for Complex Expression
To create your expression constructs (see illustration 3), introduce your gene or
genes of interest - carrying any additional modifications such as purification or
reporter tags - using your method of choice (conventional restriction-ligation cloning
or SLIC) into any of the acceptor or donor vectors. You can then create acceptordonor fusions with the help of Cre recombinase. Note that you need at least one
acceptor vector if you wish to amplify the multi-gene constructs in standard
laboratory strains. Select your multi-vector multi-gene fusions by subjecting
transformed bacteria to multiple antibiotic selection on agar and/or multi-well
plates. You will then have to extract the plasmid construct from your host strains
since the expression strain will most likely be different (see chapter C.5).
Illustration 3: Schematic representation of process for generating multi-gene expression
constructs.
If, for example, your requirements for an antibiotic resistance marker change, you
can transfer entire expression cassettes (including promoters and terminators) from
acceptor to acceptor or donor to donor by employing the homing endonucleaseBstXI module. Note that you cannot move cassettes from acceptors to donors or
ACEMBL MultiColi
June 2011
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vice versa since their homing endonuclease and BstXI recognition sites are
incompatible.
C.5. Complex Expression
For expression in E.coli, the ACEMBL multi-gene expression vector fusions with
appropriate promoters or terminators are transformed into the appropriate
expression host of choice. In the current version (T7 and lac promoter elements),
most of the wide array of currently available expression strains can be utilized. If
particular expression strains already contain helper plasmids with DNA encoding for
chaperones, lysozyme or other factors of interest, the design of the multi-gene
fusion should ideally be such that the ACEMBL vector containing the resistance
marker that is also present on the helper plasmid is not included in multi-gene
vector construction to avoid issues with plasmid incompatibility (although this is
probably not essential).
Alternatively, the issue can be resolved by creating new versions of the
ACEMBL vectors containing resistance markers that circumvent the conflict. This can
be easily performed by PCR amplifying the vectors minus the resistance marker, and
combine the resulting fragments with a PCR amplified resistance marker by
recombination (SLIC) or blunt-end ligation (using 5’phosphorylated primers). Note
that resistance markers can also be exchanged in between ACEMBL vectors by
restriction digestion with AlwNI and ClaI (for Donors) and AlwNI and PmeI (for
Acceptors).
Donor vectors depend on the pir gene product expressed by the host, due to
the R6Kγ conditional origin of replication. In regular expression strains, they rely on
fusion with an Acceptor for productive replication. Donors or Donor-Donor fusions
can nonetheless be used even for expression when not fused with an Acceptor, by
using expression strains carrying a genomic insertion of the pir gene. Such strains
have recently become available (e.g. from Novagen Inc., Madison WI, USA).
Co-transformation of two plasmids can also lead to successful protein
complex expression. The ACEMBL system contains two Acceptor vectors, pACE and
ACEMBL MultiColi
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pACE2, which are identical except for the resistance marker (Illustration 1).
Therefore, genes present on pACE1 or pACE2, respectively, can be expressed by cotransformation of the two plasmids and subsequent simultaneous exposure to
tetracyclin and ampicillin. In fact, entire Acceptor-Donor fusions containing several
genes, based on pACE1 or pACE2 as Acceptors, can in principle be co-transformed
for multi-expression, if needed.
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D. Procedures
D.1. Cloning into ACEMBL vectors
All Donors and Acceptors contain an identical MIE with exception of the homing
endonuclease site / BstXI tandem than flanks the MIE (Illustrations 1 and 3, plasmid
maps in the appendix). The MIE is tailored for sequence and ligation independent
gene insertion methods. In addition, the MIE also contains a series of unique
restriction sites, and therefore can be used as a classical polylinker for conventional
gene insertion by restriction/ligation. We suggest to choose the methods a user lab
is most familiar with. For automated applications, restriction/ligation is essentially
ruled out. In this case, recombination approaches can be used efficiently for gene
insertion (SLIC).
Illustration 4: The MIE and other cloning and multiplication elements in the Acceptor and
Donor vectors.
D.1.1. Single gene insertion into the MIE by SLIC
Several procedures for restriction/ligation independent insertion of genes into
vectors have been published or commercialized (Novagen LIC, Becton-Dickinson BD
In-Fusion and others), each with its own merit. All of these systems rely on the
exonuclease activity of DNA polymerases. In the absence of dNTPs, 5’ extensions are
created from blunt ends or overhangs by digestion from the 3’ end. If two DNA
fragments contain the same approx. 20 bp sequence at their termini at opposite
ACEMBL MultiColi
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ends, this results in overhangs that share complementary sequences capable of
annealing. This can be exploited for ligation independent combination of two or
several DNA fragments containing homologous sequences.
If T4 DNA polymerase is used, this can be carried out in a manner that is
independent of the sequences of the homology regions (Sequence and Ligation
Independent Cloning, SLIC) and detailed protocols have become available. In the
context of multi-protein expression, this is particularly useful, as the presence of
unique restriction sites, or their creation by mutagenesis, in the ensemble of
encoding DNAs ceases to be an issue.
We adapted SLIC for the insertion of encoding DNAs amplified by Phusion
polymerase into the ACEMBL Acceptor and Donor vectors according to the
published protocols. This not only allows seamless integration of genes into the
expression cassettes, but also concatamerization of expression cassettes into multigene constructs via a simple and repetitive routine that can be readily automated.
Illustration 4: Single gene insertion by SLIC. A gene of interest (GOI 1) is PCR
amplified with specific primers and integrated into a vector (Acceptor, Donor)
linearized by PCR with complementary primers (complementary regions are
shaded in light gray or dark grey, respectively). Resulting PCR fragments contain
homology regions at their ends. T4 DNA polymerase acts as an exonuclease in the
absence of dNTP and produces long sticky overhangs. Mixing (optionally
annealing) of T4DNA polymerase exonuclease treated insert and vector is
followed by transformation, yielding a single gene expression cassette.
ACEMBL MultiColi
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We use an improved protocol for SLIC which was modified from the original
publication12. This protocol, as applied manually, is detailed below (Protocol 1). If
other
systems
are
used
(BD-InFusion
etc.),
follow
the
manufacturer’s
recommendations. For robotics applications, modifications of the protocol may be
necessary and will be detailed elsewhere13.
Protocol 1: Single gene insertion by SLIC.
Reagents required:
Phusion Polymerase
5x HF Buffer for Phusion Polymerase
dNTP mix (10 mM)
T4 DNA polymerase (and10x Buffer)
DpnI enzyme
E. coli competent cells
100mM DTT, 2M Urea, 500 mM EDTA
Antibiotics
Step 1: Primer design
Primers for the SLIC procedure are designed to provide the regions of homology
that result in long sticky ends after treatment with T4 DNA polymerase in the
absence of dNTP:
Primers for the insert contain a DNA sequence corresponding to this region of
homology (“Adaptor sequence” in Illustration 4, inset), followed by a sequence
stretch that specifically anneals to the insert to be amplified (Illustration 4,
inset). Useful adaptor sequences for SLIC are listed below (Table I).
If the gene of interest (GOI) is amplified from a vector already containing
expression elements (e.g. the pET vector series), this “insert specific sequence”
can be located upstream of a ribosome binding site (rbs). Otherwise, the
forward primer needs to be designed such that a ribosome binding site is also
provided in the final construct (Illustration 4, inset).
12
13
Li and Elledge, Nat. Methods 4, 251 (2007)
Bieniossek, Nie et al., Nat. Methods 6, 447 (2009).
ACEMBL MultiColi
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Primers for PCR linearization of the vector backbone are simply complementary
to the two adaptor sequences present in the primer pair chosen for insert
amplification (Illustration 4).
Step 2: PCR amplification of insert and vector
Identical reactions are prepared in 100-µl volume for DNA insert to be cloned
and vector to be linearized by PCR:
ddH2O
5 Phusion HF Reaction buffer
dNTPs (10 mM stock)
Template DNA (100 ng/µl)
5’ SLIC primer (100 µM stock)
3’ SLIC primer (100 µM stock)
Phusion polymerase (2 U/µl)
75 µl
20 µl
2 µl
1 µl
1 µl
1 µl
0.5 µl
PCR reactions are then carried out with a standard PCR program (unless very
long DNAs are amplified, in which case you should the double extension time):
1 x 98 C for 2 min
30 x [98 C for 20 sec. -> 50C for 30 sec. -> 72C for 3 min]
Hold at 10C
Analysis of the PCR reactions by agarose gel electrophoresis and ethidium
bromide staining is recommended.
Step 3: DpnI treatment of PCR products (optional)
PCR reactions are then supplied with 1 µl DpnI enzyme which cleaves parental
plasmids (that are methylated). For insert PCR reactions, DpnI treatment is not
required if the resistance marker of the template plasmid differs from the
destination vector.
Reactions are carried out as follows:
Incubation: 37C for 1-4h
Inactivation: 80C for 20 min
Step 4: Purification of PCR products
! PCR products must be purified to remove residual dNTPs!
Otherwise, the T4 DNA polymerase reaction (Step 5) may be compromised.
Product purification is best performed by using commercial PCR Purification Kits
or NucleoSpin Kits (Qiagen, MacheryNagel or others). It is recommended to
perform elution in the minimal possible volume indicated by the manufacturer.
Step 5: T4 DNA polymerase exonuclease treatment
ACEMBL MultiColi
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Identical reactions are prepared in 20-µl volume for insert and for vector (eluted
in Step 4):
10x T4 DNA polymerase buffer
100mM DTT
2M Urea
DNA eluate from Step 3 (vector or insert)
T4 DNA polymerase
2 µl
1 µl
2 µl
14 µl
1 µl
Reactions are then carried out as follows:
Incubation: 23C for 20 min
Arrest:
Addition of 1 µl 500 mM EDTA
Inactivation: 75C for 20 min
Step 6: Mixing and Annealing
Exonuclease treated insert and vector are then mixed, followed by an (optional)
annealing step which was found to enhance efficiency14:
T4 DNA pol treated insert:
T4 DNA pol treated vector:
Annealing:
Cooling:
10 µl
10 µl
65C for 10 min
Slowly (in heat block) to RT
Step 7: Transformation
Mixtures are next transformed into competent cells following standard
transformation procedures.
Reactions for pACE1 and pACE2 derivatives are transformed into standard E. coli
cells for cloning (such as TOP10, DH5, HB101) and after recovery (2-4h) plated
on agar containing ampicillin (100 µg/ml) or tetracycline (25 µg/ml), respectively.
Reactions for Donor derivatives are transformed into E. coli cells expressing the
pir gene (such as BW23473, BW23474, or PIR1 and PIR2, Invitrogen) and plated
on agar containing chloramphenicol (25 µg/ml, pDC), kanamycin (50 µg/ml,
pDK), and spectinomycin (50 µg/ml, pDS).
Step 8: Plasmid analysis
Plasmids are cultured in small-scale in media containing the corresponding
antibiotic, and analyzed by sequencing and (optionally) restriction mapping with
an appropriate restriction enzyme.
14
Dr. Rolf Jaussi, PSI Villigen, personal communication
ACEMBL MultiColi
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D.1.2. Polycistron assembly in MIE by SLIC
The multiple integration element can also be used to integrate genes of interest by
using multi-fragment SLIC recombination as shown in Illustration 4. Genes preceded
by ribosome binding sites (rbs) can be assembled into polycistrons in this way.
Illustration 5: Generating a polycistron by SLIC. Genes of interest (GOI 1,2,3) are PCR
amplified with specific primers and integrated into a vector (Acceptor, Donor) linearized
by PCR with primers complementary to the ends of the forward primer of the first (GOI 1)
and the reverse primer of the last (GOI 3) gene to be assembled in the polycistron
(complementary regions are shaded in light gray or dark grey, respectively). Resulting PCR
fragments contain homology regions at the ends. T4 DNA polymerase acts as an
exonuclease in the absence of dNTP and produces long sticky overhangs. Mixing
(optionally annealing) of T4DNA polymerase exonuclease treated insert and vector is
followed by transformation, yielding a polycistronic expression cassette.
Protocol 2. Polycistron assembly by SLIC.
Reagents required:
Phusion Polymerase
5x HF Buffer for Phusion Polymerase
dNTP mix (10 mM)
T4 DNA polymerase (and 10x Buffer)
E. coli competent cells
ACEMBL MultiColi
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100mM DTT, 2M Urea, 500 mM EDTA
Antibiotics
Step 1: Primer design
The MIE element is composed of tried-and-tested primer sequences. These
constitute the “Adaptor” sequences that can be used for inserting single genes
or multi-gene constructs. Recommended adaptor sequences are listed below
(Table I).
Adaptor sequences form the 5’ segments of the primers used to amplify DNA
fragments to be inserted into the MIE. Insert specific sequences are added at 3’,
DNA encoding a ribosome binding site can be inserted optionally if not already
present on the PCR template
Step 2: PCR amplification of insert and primer
Identical reactions are prepared in 100-µl volume for all DNA insert (GOI 1,2,3)
to be cloned and the vector to be linearized by PCR:
ddH2O
5 Phusion HF Reaction buffer
dNTPs (10 mM stock)
Template DNA (100 ng/µl)
5’ SLIC primer (100 µM stock)
3’ SLIC primer (100 µM stock)
Phusion polymerase (2 U/µl)
75 µl
20 µl
2 µl
1 µl
1 µl
1 µl
0.5 µl
PCR reactions are then carried out with a standard PCR program (unless very
long DNAs are amplified, in which case you should double the extension time):
1 x 98 C for 2 min
30 x [98 C for 20 sec. -> 50C for 30 sec. -> 72C for 3 min]
Hold at 10C
Analysis of the PCR reactions by agarose gel electrophoresis and ethidium
bromide staining is recommended.
Step 3: DpnI treatment of PCR products (optional)
PCR reactions are then supplied with 1 µl DpnI enzyme which cleaves parental
plasmids (that are methylated). For insert PCR reactions, DpnI treatment is not
required if the resistance marker of the template plasmids differs from the
destination vector.
ACEMBL MultiColi
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Reactions are then carried out as follows:
Incubation: 37C for 1-4h
Inactivation: 80C for 20 min
Step 4: Purification of PCR products
! PCR products must be purified to remove residual dNTPs !
Otherwise, the T4 DNA polymerase reaction (Step 5) may be compromised.
Product purification is best performed by using commercial PCR Purification Kits
or NucleoSpin Kits (Qiagen, MacheryNagel or others). It is recommended to
perform elution in the minimal possible volume indicated by the manufacturer.
Step 5: T4 DNA polymerase exonuclease treatment
Identical reactions are prepared in 20-µl volume for each insert (GOI 1,2,3) and
for the vector (eluted in Step 4):
10x T4 DNA polymerase buffer
100mM DTT
2M Urea
DNA eluate from Step 3 (vector or insert)
T4 DNA polymerase
2 µl
1 µl
2 µl
14 µl
1 µl
Reactions are then carried out as follows:
Incubation: 23C for 20 min
Arrest:
Addition of 1 µl 500 mM EDTA
Inactivation: 75C for 20 min
Step 6: Mixing and Annealing
T4 DNA polymerase exonuclease treated insert and vector are then mixed,
followed by an (optional) annealing step which was found to enhance
efficiency15:
T4 DNA pol treated insert 1 (GOI 1):
T4 DNA pol treated insert 2 (GOI 2):
T4 DNA pol treated insert 3 (GOI 3):
T4 DNA pol treated vector:
Annealing:
Cooling:
5 µl
5 µl
5 µl
5 µl
65C for 10 min
Slowly (in heat block) to RT
Step 7: Transformation
Mixtures are next transformed into competent cells following standard
transformation procedures.
15
Dr. Rolf Jaussi, PSI Villigen, personal communication.
ACEMBL MultiColi
June 2011
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Reactions for pACE1 and pACE2 derivatives are transformed into standard E. coli
cells for cloning (such as TOP10, DH5, HB101) and after recovery plated on agar
containing ampicillin (100 µg/ml) or tetracycline (25 µg/ml), respectively.
Reactions for Donor derivatives are transformed into E. coli cells expressing the
pir gene (such as BW23473, BW23474, or PIR1 and PIR2, Invitrogen) and plated
on agar containing chloramphenicol (25 µg/ml, pDC), kanamycin (50 µg/ml,
pDK), and spectinomycin (50 µg/ml, pDS).
Step 8: Plasmid analysis
Plasmids are cultured and correct clones are selected based on specific
restriction digestion and DNA sequencing of the inserts.
Table I. Adaptor sequences
For single gene or multi-gene insertions into ACEMBL vectors by SLIC.
Adaptor1
Sequence
Description
T7InsFor
TAGGTATCGATAATAC
GACTCACTATAGGG
Forward primer for insert amplification, if gene
of interest (GOI) is present in a T7 system vector
(i.e. pET series).
No further extension (rbs, insert specific overlap)
required.
T7InsRev
T7VecFor
T7VecRev
CCTCAAGACCCGTTTA
GAGGCCCCAAGGGGT
TATGCTAG
Reverse primer for insert amplification, if GOI is
present in a T7 system vector (i.e. pET series).
CTAGCATAACCCCTTG
GGGCCTCTAAACGGGT
CTTGAGG
Forward primer for vector amplification, reverse
complement of T7InsRev.
CCCTATAGTGAGTCGT
ATTATCGATACCTA
Reverse primer for vector amplification, reverse
complement of T7InsFor.
No further extension (stop codon, insert specific
overlap) required.
No further extension required.
No further extension required.
NdeInsFor
GTTTAACTTTAAGAAG
GAGATATACATATG
Forward primer for insert amplification for
insertion into MIE site I1 (Illustration 2).
Further extension at 3’ (insert specific overlap)
required.
Can be used with adaptor XhoInsRev in case of
single fragment SLIC (Illustr. 3).
XhoInsRev
XhoVecFor
ACEMBL MultiColi
pACE1,pACE2,pDC
(T7):
CTTTGTTAGCAGCCGG
ATCTCTCGAG
Reverse primer for insert amplification for
insertion into MIE site I4 (Illustr. 2).
pDK,pDS (lac):
GGGTTTAAACGGAACT
AGTCTCGAG
Can be used with adaptor NdeInsFor in case of
single fragment SLIC (Illustr. 3).
pACE1,pACE2,pDC
(T7):
Forward primer for vector amplification, reverse
complement of XhoInsRev
Further extension at 3’ (stop codon, insert
specific overlap) required.
June 2011
21
CTCGAGAGATCCGGCT
GCTAACAAAG
No further extension required.
pDK,pDS (lac):
CTCGAGACTAGTTCCGT
TTAAACCC
NdeVecRev
CATATGTATATCTCCTT
CTTAAAGTTAAAC
Reverse primer for vector amplification, reverse
complement of NdeInsFor.
No further extension required.
SmaBam
GAATTCACTGGCCGTC
GTTTTACAGGATCC
Reverse primer for insert amplification (GOI1) for
insertion into MIE site I1 (Illustr. 2).
Further extension at 3’ (stop codon, insert specific
overlap) required.
Use with adaptor NdeInsFor.
BamSma
GGATCCTGTAAAACGA
CGGCCAGTGAATTC
Forward primer for insert amplification (GOI2) for
insertion into site I2 (Illustr. 2,4).
Further extension at 3’ (rbs, insert specific
overlap) required.
Use with adaptor SacHind (multifragment SLIC,
Illustr. 4)
SacHind
GCTCGACTGGGAAA
ACCCTGGCGAAGCT
T
Reverse primer for insert amplification (GOI2)
insertion into MIE site I2 (Illustr. 2, 4).
Further extension at 3’ (stop codon, insert specific
overlap) required.
Use with adaptor BamSma (multifragment SLIC,
Illustr. 4)
HindSac
AAGCTTCGCCAGGG
TTTTCCCAGTCGAGC
Forward primer for insert amplification (GOI3) for
insertion into site I3 (Illustr. 2,4).
Further extension at 3’ (rbs, insert specific
overlap) required.
Use with adaptor BspEco (multifragment SLIC,
Illustr. 4)
BspEco5
GATCCGGATGTGAA
ATTGTTATCCGCTGG
TACC
Reverse primer for insert amplification (GOI3)
insertion into MIE site I3 (Illustr. 2, 4).
Further extension at 3’ (stop codon, insert specific
overlap) required.
Use with adaptor HindSac.(multifragment SLIC,
Illustr. 4)
Eco5Bsp
GGTACCAGCGGATA
ACAATTTCACATCCG
GATC
Forward primer for insert amplification (GOI4) for
insertion into site I4 (Illustr. 2,4).
Further extension at 3’ (rbs, insert specific
overlap) required.
Use with adaptor XhoInsRev (multifragment SLIC,
Illustr. 4)
1
All Adaptor primers (without extension) can be used as sequencing primers for
genes of interest that were inserted into the MIE.
ACEMBL MultiColi
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D.1.3. Gene insertion by restriction/ligation
The MIE can also be interpreted as a simple multiple cloning site with a series of
unique restriction sites. The MIE is preceded by a promoter and a ribosome binding
site, and followed by a terminator. Therefore, cloning into the MIE by classical
restriction/ligation also yields functional expression cassettes.
Genes of interest can be subcloned by using standard cloning procedures
into the multiple integration element (MIE) (see Appendix) of ACEMBL vectors (the
MIE is identical in all vectors).
Protocol 3. Restriction/ligation cloning into the MIE.
Reagents required:
Phusion Polymerase
5x HF Buffer for Phusion Polymerase
dNTP mix (10 mM)
10 mM BSA
Restriction endonucleases (and 10x Buffer)
T4 DNA ligase (and 10x Buffer)
Calf or Shrimp intestinal alkaline phosphatase
E. coli competent cells
Antibiotics
Step 1: Primer design
For conventional cloning, PCR primers are designed containing chosen restriction
sites, preceded by appropriate overhangs for efficient cutting (c.f. New England
Biolabs catalog), and followed by ≥20 nucleotides overlapping with the gene of
interest that is to be inserted.
All MIEs are identical in the ACEMBL vectors. They contain a ribosome binding
site preceding the NdeI site. For single gene insertions, therefore, a rbs does not
need be included in the primer.
If multi-gene insertions are planned (for example into insertion sites I1-I4 of the
MIE), primers need to be designed such that a rbs preceding the gene and a stop
codon at its end are provided.
In particular for polycistron cloning by restriction/ligation, it is recommended to
construct templates by custom gene synthesis. In the process, the restriction
sites present in the MIE can be eliminated from the encoding DNAs.
ACEMBL MultiColi
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Step 2: Insert preparation
PCR of insert(s):
Identical PCR reactions are prepared in 100 µl volume for genes of interest to be
inserted into the MIE:
ddH2O
5 Phusion HF Reaction buffer
dNTPs (10 mM stock)
Template DNA (100 ng/µl)
5’ primer (100 µM stock)
3’ primer (100 µM stock)
Phusion polymerase (2 U/µl)
75 µl
20 µl
2 µl
1 µl
1 µl
1 µl
0.5 µl
PCR reactions are then carried out with a standard PCR program (unless very
long DNAs are amplified, then double extension time):
1 x 98 C for 2 min
30 x [98 C for 20 sec. -> 50C for 30 sec. -> 72C for 3 min]
Hold at 10C
Analysis of the PCR reactions by agarose gel electrophoresis and ethidium
bromide staining is recommended.
Product purification is best performed by using commercial PCR Purification Kits
or NucleoSpin Kits (Qiagen, MacheryNagel or others). It is recommended to
perform elution in the minimal possible volume indicated by the manufacturer.
Restriction digestion of insert(s):
Restriction reactions are carried out in 40 µl reaction volumes, using the specific
restriction enzymes as specified by the manufacturer’s recommendations (c.f.
New England Biolabs catalog and others).
PCR Kit eluate (≥1 µg)
10x Restriction enzyme buffer
10 mM BSA
Restriction enzyme for 5’
Restriction enzyme for 3’
30 µl
4 µl
2 µl
2 µl
2 µl (in case of double digestion, else
ddH2O)
Restriction digestions are performed in a single reaction with both enzymes
(double digestion) or sequentially (two single digestions) if the buffer conditions
required are incompatible.
Gel extraction of insert(s):
ACEMBL MultiColi
June 2011
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Processed insert is then purified by agarose gel extraction using commercial kits
(Qiagen, MachereyNagel etc). It is recommended to elute the extracted DNA in
the minimal volume defined by the manufacturer.
Step 3: Vector preparation
Restriction digestion of ACEMBL plasmid(s):
Restriction reactions are carried out in 40 µl reaction volumes, using specific
restriction enzymes as specified by manufacturer’s recommendations (c.f. New
England Biolabs catalog and others).
ACEMBL plasmid (≥0.5 µg) in ddH2O
10x Restriction enzyme buffer
10 mM BSA
Restriction enzyme for 5’
Restriction enzyme for 3’
30 µl
4 µl
2 µl
2 µl
2 µl (for double digestions, else
ddH2O)
Restriction digestions are performed in a single reaction with both enzymes
(double digestion) or sequentially (two single digestions) if the buffer conditions
required are incompatible.
Gel extraction of vector(s):
Processed vector is then purified by agarose gel extraction using commercial
kits (Qiagen, MachereyNagel etc). It is recommended to elute the extracted
DNA in the minimal volume defined by the manufacturer.
Step 4: Ligation
Ligation reactions are carried out in 20 µl reaction volumes according to the
recommendations of the supplier of T4 DNA ligase:
ACEMBL plasmid (gel extracted)
Insert (gel extracted)
10x T4 DNA Ligase buffer
T4 DNA Ligase
8 µl
10 µl
2 µl
0.5 µl
Ligation reactions are performed at 25°C (sticky end) for 1h or at 16°C (blunt
end) overnight.
Step 5: Transformation
Mixtures are next transformed into competent cells following standard
transformation procedures.
Reactions for pACE1 and pACE2 derivatives are transformed into standard E. coli
cells for cloning (such as TOP10, DH5, HB101) and after recovery plated on agar
containing ampicillin (100 µg/ml) or tetracycline (25 µg/ml), respectively.
Reactions for Donor derivatives are transformed into E. coli cells expressing the
pir gene (such as BW23473, BW23474, or PIR1 and PIR2, Invitrogen) and plated
ACEMBL MultiColi
June 2011
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on agar containing chloramphenicol (25 µg/ml, pDC), kanamycin (50 µg/ml,
pDK), and spectinomycin (50 µg/ml, pDS).
Step 6: Plasmid analysis
Plasmids are cultured and correct clones are selected based on specific
restriction digestion and DNA sequencing of the inserts.
D.1.4. Multiplication by using the HE and BstXI sites
All ACEMBL system vectors contain a homing endonuclease (HE) site and a
designed BstXI site that envelop the multiple integration element (MIE). The homing
endonuclease site can be used to insert entire expression cassettes, containing
single genes or polycistrons, into a vector already containing one gene or several
genes of interest. Homing endonucleases have long recognition sites (20-30 base
pairs or more). Although not all equally stringent, homing endonuclease sites are
most probably unique in the context of even large plasmids, or, in fact, entire
genomes.
In the ACEMBL system, Donor vectors contain a recognition site for homing
endonuclease PI-SceI (Illustr. 2). This HE site yields upon cleavage a 3’ overhang with
the sequence -GTGC. Acceptor vectors contain the homing endonuclease site I-CeuI,
which upon cleavage will result in a 3’ overhang of -CTAA. On Acceptors and Donors,
the respective HE site is preceding the MIE. The 3’ end of the MIE contains a
specifically designed BstXI site, which upon cleavage will generate a matching
overhang. The basis of this is the specificity of cleavage by BstXI. The recognition
sequence of BstXI is defined as CCANNNNN’NTGG (apostrophe marks position of
phosphodiester link cleavage). The residues denoted as N can be chosen freely.
Donor vectors thus contain a BstXI recognition site of the sequence
CCATGTGC’CTGG, and Acceptor vectors contain CCATCTAA’TTGG. The overhangs
generated by BstXI cleavage in each case will match the overhangs generated by HE
cleavage. Note that Acceptors and Donors have different HE sites.
The recognition sites are not symmetric. Therefore, ligation of a HE/BstXI
digested fragment into a HE site of an ACEMBL vector will be (1) directional and (2)
result in a hybrid DNA sequence where a HE halfsite is combined with a BstXI
halfsite. This site will be cut by neither HE nor BstXI. Therefore, in a construct that
ACEMBL MultiColi
June 2011
26
had been digested with a HE, insertion by ligation of HE/BstXI digested DNA
fragment containing an expression cassette with one or several genes will result in a
construct which contains all heterologous genes of interest, enveloped by an intact
HE site in front, and a BstXI site at the end. Therefore, the process of integrating
entire expression cassettes by means of HE/BstXI digestion and ligation into a HE
site can be repeated iteratively.
Protocol 4. Multiplication by using homing endonuclease/BstXI.
Reagents required:
Homing endonucleases PI-SceI, I-CeuI
10x Buffers for homing endonucleases
Restriction enzyme BstXI (and 10x Buffer)
T4 DNA ligase (and 10x Buffer)
E. coli competent cells
Antibiotics
Step 2: Insert preparation
Restriction reactions are carried out in 40 µl reaction volumes, using homing
endonucleases PI-SceI (Donors) or I-CeuI (Acceptors) as recommended by the
supplier (c.f. New England Biolabs catalog and others).
ACEMBL plasmid ( ≥ 0.5 µg) in ddH2O
10x Restriction enzyme buffer
10 mM BSA
PI-SceI (Donors) or I-CeuI (acceptors)
32 µl
4 µl
2 µl
2 µl
Reactions are then purified by PCR extraction kit or acidic ethanol precipitation,
and next digested by BstXI according to the recommendations of the supplier.
HE digested DNA in ddH2O
10x Restriction enzyme buffer
10 mM BSA
BstXI
32 µl
4 µl
2 µl
2 µl
Gel extraction of insert(s):
Processed insert is then purified by agarose gel extraction using commercial kits
(Qiagen, MachereyNagel etc). It is recommended to elute the extracted DNA in
the minimal volume defined by the manufacturer.
ACEMBL MultiColi
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Step 3: Vector preparation
Restriction reactions are carried out in 40 µl reaction volumes, using homing
endonucleases PI-SceI (Donors) or I-CeuI (Acceptors) as recommended by the
supplier (c.f. New England Biolabs catalog and others).
ACEMBL plasmid ( ≥ 0.5 µg) in ddH2O
10x Restriction enzyme buffer
10 mM BSA
PI-SceI (Donors) or I-CeuI (acceptors)
33 µl
4 µl
2 µl
1 µl
Reactions are then purified by PCR extraction kit or acidic ethanol precipitation,
and next treated with intestinal alkaline phosphatase according to the
recommendations of the supplier.
HE digested DNA in ddH2O
10x Alkaline phosphatase buffer
Alkaline phosphatase
17 µl
2 µl
1 µl
Gel extraction of vector:
Processed vector is then purified by agarose gel extraction using commercial
kits (Qiagen, MachereyNagel etc). It is recommended to elute the extracted
DNA in the minimal volume defined by the manufacturer.
Step 4: Ligation
Ligation reactions are carried out in 20 µl reaction volumes:
HE/Phosphatase treated vector (gel extracted)
HE/BstXI treated insert (gel extracted)
10x T4 DNA Ligase buffer
T4 DNA Ligase
4 µl
14 µl
2 µl
0.5 µl
Ligation reactions are performed at 25ºC for 1h or at 16ºC overnight.
Step 5: Transformation
Mixtures are next transformed into competent cells following standard
transformation procedures.
Reactions for pACE and pACE2 derivatives are transformed into standard E. coli
cells for cloning (such as TOP10, DH5, HB101) and after recovery plated on agar
containing ampicillin (100 µg/ml) or tetracycline (25 µg/ml), respectively.
Reactions for Donor derivatives are transformed into E. coli cells expressing the
pir gene (such as BW23473, BW23474, or PIR1 and PIR2, Invitrogen) and plated
on agar containing chloramphenicol (25 µg/ml, pDC), kanamycin (50 µg/ml,
pDK), and spectinomycin (50 µg/ml, pDS).
Step 6: Plasmid analysis
ACEMBL MultiColi
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Plasmids are cultured and correct clones selected based on specific restriction
digestion and DNA sequencing of the inserts.
Note: Integration can likewise be performed by sequence and ligation independent
cloning. It is recommended to carry out linearization of the vector by digestion with
HE, if heterologous genes are already present, to avoid PCR amplifications over
encoding regions. The fragment to be inserted is generated by PCR amplification
resulting in a PCR fragment containing a 20-25 base pair stretch at its 5’ end that is
identical to the corresponding DNA sequence present at the HE site counted from
the site of cleavage towards 5’ (site of cleavage is position -4). At the 3’ end of the
PCR fragment, the homology region is 20-25 base pairs counted from the site of
cleavage towards 3’.
D.2. Cre-LoxP reaction of Acceptors and Donors
Cre recombinase is a member of the integrase family (Type I topoisomerase from
bacteriophage P1). It recombines a 34 bp loxP site in the absence of accessory
protein or auxiliary DNA sequence. The loxP site is comprised of two 13 bp
recombinase-binding elements arranged as inverted repeats which flank an 8 bp
central region where cleavage and ligation reaction occur.
The site-specific recombination mediated by Cre recombinase involves the
formation of a Holliday junction (HJ). The recombination events catalyzed by Cre
recombinase are dependent on the location and relative orientation of the loxP
sites. Two DNA molecules, for example an Acceptor and a Donor plasmid, containing
single loxP sites will be fused. Furthermore, the Cre recombination is an equilibrium
reaction with 20-30% efficiency in recombination. This provides useful options for
multi-gene combinations for multi-protein complex expression.
Illustration 6: LoxP imperfect inverted repeat
13bp
5’…ATAACTTCGTATA
3’…TATTGAAGCATAT
inverted repeat
8bp
13bp
GCATACAT TATACGAAGTTAT…3’
CGTATGTA ATATGCTTCAATA…5’
Spacer
inverted repeat
In a reaction where several DNA molecules such as Donors and Acceptors are
incubated with Cre recombinase, the fusion/excision activity of the enzyme will
result in an equilibrium state where single vectors (educt vectors) and all possible
fusions coexist. Donor vectors can be used with Acceptors and/or Donors, likewise
ACEMBL MultiColi
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for Acceptor vectors. Higher order fusions are also generated where more than two
vectors are fused. This is shown schematically in Illustration 6.
The fact that Donors contain a conditional origin of replication that depends
on a pir+ (pir positive) background now allows for selecting out from this reaction
mix all desired Acceptor-Donor(s) combinations. For this, the reaction mix is used to
transform to pir negative strains (TOP10, DH5, HB101 or other common laboratory
cloning strains). Then, Donor vectors will act as suicide vectors when plated out on
agar containing the antibiotic corresponding to the Donor encoded resistance
marker, unless fused with an Acceptor. By using agar with the appropriate
combinations of antibiotics, all desired Acceptor-Donor fusions can be selected for.
Using this approach, fusion vectors of 25 kb and larger have generated. In
stability tests (serial passaging for more than 60 generations), even such large
plasmids proved to be stable as checked by restriction mapping, even if only one of
the antibiotics corresponding to the encoded resistance markers was provided in
the growth medium.
ACEMBL MultiColi
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Illustration 7: Cre and De-Cre reaction pyramid
Cre-mediated assembly and disassembly of pACE, pDK, and pDS vectors are shown in a
schematic representation (left side). LoxP sites are shown as red circles, resistance markers
and origins are labeled. White arrows stand for the entire expression cassette (including
promoter, terminator and multiple integration elements) in the ACEMBL vectors. Not all
possible fusion products are shown for clarity. Levels of multi-resistance are indicated
(right).
D.2.1. Cre-LoxP fusion of Acceptors and Donors
This protocol is designed for generating multi-gene fusions from Donors and
Acceptors by Cre-LoxP reaction.
Reagents:
Cre recombinase (from NEB or self made)
Standard E. coli competent cells (pir- strain)
Antibiotics
96well microtiter plates
12 well tissue-culture plates (or petri dishes) w. agar/antibiotics
ACEMBL MultiColi
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LB medium
1. For a 20µl Cre reaction, mix 1-2 µg of each educt in approximately equal
amounts. Add ddH2O to adjust the total volume to 16-17 µl, then add 2 µl
10x Cre buffer and 1-2µl Cre recombinase.
2. Incubate Cre reaction at 37°C (or 30°C) for 1 hour.
3. Optional: load 2-5 µl of Cre reaction on an analytical agarose gel for
examination.
Heat inactivation at 70°C for 10 minutes before the gel loading is strongly
recommended.
4. For chemical transformation, mix 10-15µl Cre reaction with 200 µl chemical
competent cells. Incubate the mixture on ice for 15-30 minutes. Then
perform heat shock at 42°C for 45-60 s.
Up to 20 µl Cre reaction (0.1 volumes of the chemical competent cell suspension) can
be directly transformed into 200 µl chemical competent cells.
For electrotransformation, up to 2 µl Cre reaction could be directly mixed
with 100 µl electrocompetent cells, and transformed by using an
electroporator (e.g. BIORAD E. coli Pulser) at 1.8-2.0 kV.
Larger volume of Cre reaction must be desalted by ethanol precipitation or PCR
purification column before electrotransformation. The desalted Cre reaction mix
should not exceed 0.1 volumes of the electrocompetent cell suspension.
The cell/DNA mixture could be immediately used for electrotransformation without
prolonged incubation on ice.
5. Add up to 400 µl of LB media (or SOC media) per 100 µl of cell/DNA
suspension immediately after the transformation (heat shock or
electroporation).
6. Incubate the suspension in a 37°C shaking incubator overnight or for at least
4 hours (recovery period).
For recovering multifusion plasmid containing more than 2 resistance markers, it is
strongly recommended to incubate the suspension at 37°C overnight.
7. Plate out the recovered cell suspension on agar containing the desired
combination of antibiotics. Incubate at 37°C overnight.
8. Clones from colonies present after overnight incubation can be verified by
restriction digestion at this stage (refer to steps 12-16).
Especially in the case that only one multifusion plasmid is desired.
For further selection by single antibiotic challenges on a 96 well microtiter
plate, continue to step 9.
ACEMBL MultiColi
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Several to many different multifusion plasmid combinations can be processed and
selected on one 96 well microtiter plate in parallel.
9. For 96 well antibiotic tests, inoculate four colonies from each agar plate with
different antibiotic combination into ~500 µl LB media without antibiotics.
Incubate the cell cultures in a 37°C shaking incubator for 1-2 hours.
10. During the incubation of colonies, fill a 96 well microtiter plate with 150 µl
antibiotic-containing LB media (following Illustration 7). It is recommended
to add coloured dye (positional marker) in the wells indicated.
A typical arrangement of the solutions, which is used for parallel selections of
multifusion plasmids, is shown in Illustration 7. The concept behind the 96 well plate
experiment is that every cell suspension from single colonies needs to be challenged
by all four single antibiotics for unambiguous interpretation.
Illustration 8: 96 well analysis of Cre assembly
11. Add 1 µl aliquots of pre-incubated cell culture (Step 9) to the corresponding
wells. Then incubate the inoculated 96 well microtiter plate in a 37°C shaking
incubator overnight at 180-200 rpm.
Recommended: use parafilm to wrap the plate to avoid drying out.
The remainder of the pre-incubated cell cultures could be kept at 4°C for further
inoculations if necessary.
12. Select transformants containing desired multifusion plasmids based on
antibiotic resistance, according to the combination of dense (positive) and
clear (no growth) cell microcultures from each colony. Inoculate 10-20 µl cell
ACEMBL MultiColi
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culture into 10 ml LB media with corresponding antibiotics. Incubate in a
37°C shaking incubator overnight.
13. Centrifuge the overnight cell cultures at 4000g for 5-10 minutes. Purify
plasmid from the resulting cell pellets with common plasmid miniprep kits,
according to manufacturers’ recommendation.
14. Determine the concentrations of purified plasmid solutions by using UV
absorption spectroscopy (e.g. by using a NanoDropTM 1000 machine).
15. Digest approx. 0.5-1 µg of the purified plasmid solution in a 20 µl restriction
digestion with appropriate endonuclease(s). Incubate under recommended
reaction condition for ~2 hours.
16. Use 5-10 µl of the digestion for analytical agarose (0.8-1.2%) gel
electrophoresis. Verify plasmid integrity by comparing the experimental
restriction pattern to a restriction pattern predicted in silico (e.g. by using
program VectorNTI from Invitrogen or similar programs).
D.2.2. Deconstruction of fusion vectors by Cre
The protocol is suitable for releasing any single educt from multifusion constructs
(deconstruction). This is achieved by Cre-LoxP reaction, transformation and plating
on agar with appropriately reduced antibiotic resistance level (c.f. Illustration 6). In
the liberated educt entity, encoding genes can be modified and diversified. Then,
the diversified construct is resupplied by Cre-LoxP reaction (C.2.1.).
Reagents:
Cre recombinase (and 10x Buffer)
E. coli competent cells
(pir+ strains, pir- strains could be used only when partially deconstructed AcceptorDonor fusions are desired).
Antibiotics
1. Incubate approx. 1 µg multifusion plasmid with 2 µl 10x Cre buffer, 1-2 µl Cre
recombinase, add ddH2O to adjust the total reaction volume to 20 µl.
2. Incubate this Cre deconstruction reaction mixture at 30°C for 1-4 hour.
3. Optional: load 2-5 µl of the reaction on an analytical agarose gel for
examination.
Heat inactivation at 70°C for 10 minutes before the gel loading is strongly
recommended.
ACEMBL MultiColi
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4. For chemical transformation, mix 10-15µl De-Cre reaction with 200 µl
chemical competent cells. Incubate the mixture on ice for 15-30 minutes.
Then perform heat shock at 42°C for 45-60 s.
Up to 20 µl De-Cre reaction (0.1 volumes of the chemical competent cell suspension)
can be directly transformed into 200 µl chemical competent cells.
For electrotransformation, up to 2 µl De-Cre reaction can be directly mixed
with 100 µl electrocompetent cells, and transformed by using an
electroporator (e.g. BIORAD E. coli Pulser) at 1.8-2.0 kV.
Larger volume of De-Cre reaction must be desalted by ethanol precipitation or PCR
purification column before electrotransformation. The desalted De-Cre reaction mix
should not exceed 0.1 volumes of the electrocompetent cell suspension.
The cell/DNA mixture could be immediately used for electrotransformation without
prior incubation on ice.
5. Add up to 400 µl of LB media (or SOC media) per 100 µl of cell/DNA
suspension immediately after the transformation (heat shock or
electroporation).
6. Incubate the suspension in a 37°C shaking incubator (recovery).
For recovery of partially deconstructed double/triple fusions, incubate the
suspension in a 37°C shaking incubator for 1 to 2 hours.
For recovery of individual educts such as single ACEMBL vectors from pACKS plasmid,
incubate the suspension in a 37°C shaking incubator overnight or for at least 4 hours.
7. Plate out the recovered cell suspension on agar containing the desired
(combination of) antibiotic(s). Incubate at 37°C overnight.
8. Colonies after overnight incubation might be verified directly by restriction
digestion at this stage (refer to steps 12-16).
Especially recommended in the case that only one single educt or partially
deconstructed multifusion plasmid is desired.
For further selection by single antibiotic challenge on a 96 well microtiter
plate, continue with step 9.
Several different single educts/partially deconstructed multifusion plasmids can be
processed and selected on one 96 well microtiter plate in parallel.
9. For 96 well analysis, inoculate four colonies each from agar plates containing
a defined set of antibiotics into ~500 µl LB media without antibiotics.
Incubate the cell cultures in a 37°C shaking incubator for 1-2 hours.
10. During the incubation of colonies, fill a 96 well microtiter plate with 150 µl
antibiotic-containing LB media or coloured dye (positional marker) in the
corresponding wells.
Refer to Illustrations 7 and 12 for the arrangement of the solutions in the wells,
which are used for parallel selection of single educts or partially deconstructed
multifusion plasmids. The concept is that every cell suspension from a single colony
needs to be challenged by all four antibiotics separately for unambiguous
interpretation.
ACEMBL MultiColi
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11. Add 1 µl aliquots from the pre-incubated cell cultures (Step 9) into the
corresponding wells. Then incubate the 96 well microtiter plate in a 37°C
shaking incubator overnight at 180-200 rpm.
Recommended: use parafilm to wrap the plate to prevent desiccation.
The remainder of the pre-incubated cell cultures can be kept in 4°C fridge for further
inoculations if necessary.
12. Select transformants containing desired single educts or partially
deconstructed multifusion plasmids according to the combination of dense
(growth) and clear (no growth) cell cultures from each colony. Inoculate 1020 µl cell cultures into 10 ml LB media with corresponding antibiotic(s).
Incubate in a 37°C shaking incubator overnight.
13. Centrifuge the overnight cell cultures at 4000g for 5-10 minutes. Purify
plasmid from cell pellets with common plasmid miniprep kits, according to
manufacturers’ information.
14. Determine the concentrations of purified plasmid solutions by using UV
absorption spectroscopy (e.g. NanoDropTM 1000).
15. Digest approx. 0.5-1 µg of the purified plasmid solution in a 20 µl restriction
digestion (with 5-10 unit endonuclease). Incubate under recommended
reaction condition for ~2 hours.
16. Use 5-10 µl of the digestion for analytical agarose gel (0.8-1.2%)
electrophoresis. Verify the plasmid integrity by comparing the actual
restriction pattern to predicted restriction pattern in silico (e.g. by using
VectorNTI, Invitrogen, or any other similar program).
17. Optional: Possibly, a deconstruction reaction is not complete but yields
partially deconstructed fusions which still retain entities to be eliminated. In
this case, we recommend to pick these partially deconstructed fusions
containing and perform a second round of Cre deconstruction reaction
(repeat steps 1-8) by using this construct as starting material.
In our hands, two sequential deconstruction reactions were always sufficient to
recover all individual modules, for instance all four single ACEMBL vectors from a
pACKS plasmid. Liberation of single educts from double/triple fusions were found to
be often more efficient than from quadruples such as the pACKS plasmid of the
system kit (Section E).
D.3. Co-expression by Co-transformation
Protein complexes can be expressed also from two separate vectors co-transformed
in expression strains. The co-transformed vectors can have the same or different
origins of replication, however, they must encode different resistance markers.
ACEMBL MultiColi
June 2011
36
Plasmids pACE1 (ampicillin resistance marker) and pACE2 (tetracycline resistance
marker) have both a ColE1 derived replicon and can therefore be used with all
common expression strains. pACE1 and pACE2 derivatives (also including fused
Donors if needed) can be co-transformed into expression strains, and double
transformants selected for by plating on agar plates containing both ampicillin and
tetracyclin antibiotics.
Transformations are carried out by using standard transformation protocols.
ACEMBL MultiColi
June 2011
37
E. ACEMBL multi-gene combination: Examples
Examples of multi-protein expressions by ACEMBL illustrating the gene combination
procedures detailed in Section C are shown in the following. Reactions presented
were carried out manually following the protocols provided, and also on a Tecan
Freedom EvoII 200 robot with adapted protocols. These and other examples have
been published in Bieniossek et al., Nat. Methods 6, 447 (2009) plus supplementary
material.
E.1. SLIC cloning into ACEMBL vectors: human TFIIF
Genes encoding full-length human RAP74 with a C-terminal oligo-histidine tag and
full-length human RAP30 were amplified from pET-based plasmid template 16
following the protocols described above. Linearized vector backbones were
generated by PCR amplification from pACE1 and pDC. SLIC following Protocol 1
(Section D), yielded pACE1-RAP30 and pDC-RAP74his (Fig 8). These plasmids were
fused by Cre-LoxP reaction (Section C). Results from restriction mapping by
BstZ17I/BamHI double digestion of 11 double resistant (Cm, Ap) colonies are shown
by a gel section from 1% E-gel electrophoresis (M: NEB 1kb DNA marker). All clones
tested showed the expected pattern (5.0 + 2.8 kb). One clone was transformed in
BL21(DE3)
cells.
Expression
and
purification
by
Ni2+-capture
and
S200
chromatography resulted in human TFIIF complex.
Illustration 9: ACEMBLing TFIIF (Image courtesy of Dr. I. Berger, EMBL Grenoble).
16
Gaiser, Tan and Richmond,. J. Mol. Biol. 302, 1119 (2000).
ACEMBL MultiColi
June 2011
38
E.2. The Homing endonuclease/BstXI module: yeast RES complex
Plasmids pCDFDuet-Pml1p, pRSFDuet-Snu17p-NHis and pETDuet-Bud13p, encoding
for yeast proteins (all full-length) PmI1p, Snu17p and Bud13p, respectively, were a
kind gift from Dr. Simon Trowitzsch and Dr. Markus Wahl (MPI Göttingen). Snu17p
contains a six-histidine tag fused to its N-terminus. The gene encoding for His6tagged Snu17p was excised from pRSFDuet-Snu17p-NHis by NcoI/XhoI restriction,
and ligated into a NcoI/XhoI digested pACE1 construct (containing an unrelated gene
between the NcoI and XhoI sites) resulting in pACE1-Snu17. The gene encoding for
Bud13p was liberated from pETDuet-Bud13p by restriction digestion with XbaI and
EcoRV, and placed into XbaI/PmeI digested pDC resulting in pDC-Bud13. The gene
encoding Pm1Ip was liberated from pCDFDuet-Pml1p by restriction digestion with
NdeI and XhoI, and placed into NdeI/XhoI digested pDC resulting in pDC-PmI1. Next,
the expression cassette for Bud13p was liberated from pDC-Bud13 by digestion with
PI-SceI and BstXI. The liberated fragment was inserted into PI-SceI digested and
alkaline phosphatase treated pDC-PmI1p resulting in pDC-Bud13p-PmI1p.
pACE-Snu17 and pDC-BudPmI were then fused by Cre-LoxP reaction and
selected for by plating on agar plates containing ampicillin and chloramphenicol.
Fusion plasmids were transformed into BL21(DE3) cells. Expression and purification
by Ni2+-capture and S200 size exclusion chromatography resulted in the trimeric RES
complex.
Illustration 10: The HE/BstXI multiplication module.
ACEMBL MultiColi
June 2011
39
F. Appendix
F.1. DNA sequence of MIE
Below are the sequence and map of the MIE fragment between T7/lac promoter
and T7 terminator in ACEMBL vectors. Forward and reverse primers for sequencing
can be standard vector primers for T7 and lac. Adaptor primer sequences (c.f. Table I)
are indicated. DNA sequences in these homology regions contain tried-and-tested
sequencing primers17. Sites of insertion (I1-I4) are shown.. The adaptor sequences,
and probably any sequence in the homology regions, can be used as adaptors for
multifragment insertions. The ribosome binding site present in the MIE (rbs) is
boxed in red.
17
Tan S et al. Protein Expr. Purif. 40, 385 (2005).
ACEMBL MultiColi
June 2011
40
F.2. DNA sequences of ACEMBL vectors
Below are the complete sequences of all acceptor and donor vectors.
Please note though that copy-pasting from a PDF may result in faulty data transfer into
other applications, i.e. DNA sequences may lack base pairs.
To avoid any trouble, simply request the vector sequence data at [email protected] We will provide them either in text format or as ApE files that will give you
ample annotation of the features contained on the vectors. The ApE plasmid editor can be
downloaded for free from its author’s web site (M. Wayne Davis, University of Utah).
ACEMBL MultiColi
June 2011
41
F.2.1. pACE1
1
51
101
151
201
251
301
351
401
451
501
551
601
651
701
751
801
851
901
951
1001
1051
1101
1151
1201
1251
1301
1351
1401
1451
1501
1551
1601
1651
1701
1751
1801
1851
1901
1951
2001
2051
2101
2151
2201
2251
2301
2351
2401
2451
2501
2551
2601
2651
GGTACCGCGG
TAGTACTTCG
CGGTCCTAAG
AATTGTGAGC
AAGAAGGAGA
TGAATTCCCC
GGTACCAGCG
GATCCGGCTG
CGCTGAGCAA
GGGGTTTTTT
AGCGGGCTTT
AAATATGTAT
ATTGAAAAAG
CCCTTTTTTG
CGTGAAAGTA
TAGAACTGGA
GAGCGTTTTC
ATTATCCCGT
ATTCGCAGAA
ACGGACGGAA
TGATAACACT
AATTAACCGC
CGTTGGGAAC
CACAATGCCT
AACTACTTAC
GATAAAGTTG
TATTGCTGAT
CCGCACTTGG
ACGGGTAGTC
AGGGGCTTCA
TTTGGAGCCT
CTTGAGATCC
CCACCGCTAC
TTTTCCGAAG
TTCTAGTGTA
CCTACATACC
CGATAAGTCG
AGGCGCAGCG
GAGCGAACGA
AAGCGCCACG
GCAGGGTCGG
TGGTATCTTT
ATTTTTGTGA
ACGCGGCCTT
TTCTTTCCTG
TGAGTGAGCT
CAGTGAGCGA
CATCTGTGCG
TCTGATGCCG
TGGGTCATGG
ACGGGCTTGT
CCGGGAGCTG
AGGCAGGGGG
AT
ACEMBL MultiColi
CCGCGTAGAG
TTAATACAGA
GTAGCGACCT
GGATAACAAT
TATACATATG
GGGAAGCTTC
GATAACAATT
CTAACAAAGC
TAACTAGCAT
GGTTTAAACC
TTTTTAATTC
CCGCTCATGA
GAAGAGTATG
CGGCATTTTG
AAAGACGCAG
CTTGAATAGC
CAATGATGAG
GTAGATGCGG
TGACTTGGTT
TGACGGTAAG
GCGGCGAACT
TTTTTTGCAC
CAGAACTGAA
GCGGCAATGG
TCTGGCTTCA
CAGGACCACT
AAATCTGGGG
ACCAGATGGT
AGGCAACTAT
CTGATTAAGC
TTTTTTTTGG
TTTTTTTCTG
CAGCGGTGGT
GTAACTGGCT
GCCGTAGTTA
TCGCTCTGCT
TGTCTTACCG
GTCGGGCTGA
CCTACACCGA
CTTCCCGAAG
AACAGGAGAG
ATAGTCCTGT
TGCTCGTCAG
TTTACGGTTC
CGTTATCCCC
GATACCGCTC
GGAAGCGGAA
GTATTTCACA
CATAGTTAAG
CTGCGCCCCG
CTGCTCCCGG
CATGTGTCAG
AATTCCAGAT
GATCTGTTGA
TGTAGGTGTT
AGGTATCGAT
TCCCCTCTAG
AGGCCTCGGA
GCCAGGGTTT
TCACATCCGG
CCGAAAGGAA
AACCCCTTGG
CATCTAATTG
CCCTATTTGT
GACAATAACC
AGTATTCAAC
CCTTCCTGTT
AGGACCAATT
GGTAAAATCC
CACTTTCAAA
GGCAAGAGCA
GAATACTCCC
AGAATTATGT
TACTTCTGAC
AATATGGGAG
TGAAGCCATA
CAACAACATT
CGGCAACAAT
ACTGCGTTCG
CAGGAGAGCG
AAGCCTTCCC
GGACGAACGA
ATTGGTAAAC
ACGGACCGGT
CGCGTAATCT
TTGTTTGCCG
TCAGCAGAGC
GGCCACCACT
AATCCTGTTA
GGTTGGACTC
ACGGGGGGTT
ACTGAGATAC
GGAGAAAGGC
CGCACGAGGG
CGGGTTTCGC
GGGGGCGGAG
CTGGCCTTTT
TGATTCTGTG
GCCGCAGCCG
GAGCGCCTGA
CCGCAATGGT
CCAGTATACA
ACACCCGCCA
CATCCGCTTA
AGGTTTTCAC
AACTTCGTAT
TCAGCAGTTC
GGCACCATGC
AATACGACTC
AAATAATTTT
TCCTGTAAAA
TCCCAGTCGA
ATCGCGAACG
GCTGAGTTGG
GGCCTCTAAA
GACTAGTAGC
TTATTTTTCT
CTGATAAATG
ATTTCCGTGT
TTTGCTCACC
GGGGGCACGA
TTGAGAGTTT
GTTCTGCTAT
ACTCGGACGA
CAGTGACAGA
AGTGCCGCCA
AACCATCGGT
ACCATGTAAC
CCAAACGACG
ACGCAAACTA
TAATAGACTG
GCACTTCCTG
TGGTTCACGG
GTATCGTAGT
AATAGACAGA
CGATACAATT
AGAAAAGATC
GCTGCTTGCA
GATCAAGAGC
GCAGATACCA
TCAAGAACTC
CCAGTGGCTG
AAGACGATAG
CGTGCACACA
CTACAGCGTG
GGACAGGTAT
AGCTTCCAGG
CACCTCTGAC
CCTATGGAAA
GCTGGCCTTT
GATAACCGTA
AACGACCGAG
TGCGGTATTT
GCACTCTCAG
CTCCGCTATC
ACACCCGCTG
CAGACAAGCT
CGTCATCACC
AATGTATGCT
AACCTGTTGA
ATAACTATAA
ACTATAGGGG
GTTTAACTTT
CGACGGCCAG
GCTCGATATC
CGTCTCGAGA
CTGCTGCCAC
CGGGTCTTGA
CCGCCTAATG
AAATACATTC
CTTCAATAAT
CGCCCTTATT
CAGAAACGCT
GTGGGATACA
TCGCCCTGAA
GTGGAGCAGT
CGAATACACT
AAAGCACCTT
TAACGATGAG
GGACCGAAGG
TCGCCTTGAC
AGCGAGACAC
TTAACTGGCG
GCTTGAAGCG
CTGGCTGGTT
GGTATCATTG
TATCTACACG
TTGCTGAAAT
AAAGGCTCCT
AAAGGATCTT
AACAAAAAAA
TACCAACTCT
AATACTGTCC
TGTAGCACCG
CTGCCAGTGG
TTACCGGATA
GCCCAGCTTG
AGCTATGAGA
CCGGTAAGCG
GGGAAACGCC
TTGAGCGTCG
AACGCCAGCA
TGCTCACATG
TTACCGCCTT
CGCAGCGAGT
TCTCCTTACG
TACAATCTGC
GCTACGTGAC
ACGCGCCCTG
GTGACCGTCT
GAAACGCGCG
ATACGAAGTT
June 2011
42
F.2.2. pACE2
1
51
101
151
201
251
301
351
401
451
501
551
601
651
701
751
801
851
901
951
1001
1051
1101
1151
1201
1251
1301
1351
1401
1451
1501
1551
1601
1651
1701
1751
1801
1851
1901
1951
2001
2051
2101
2151
2201
2251
2301
2351
2401
2451
2501
2551
2601
2651
2701
2751
2801
2851
2901
2951
ATGAAATCTA
TGCTGTAGGC
ATATCGTCCA
CTATATGCGT
CGACCGCTTT
CTATCGACTA
GCCGGACGCA
CGCCTATATC
GGCTCATGAG
GGGGGACTGT
GGTGCTCAAC
CGCATAAGGG
AGCTCCTTCC
TGTCTTCTTT
TCATTTTCGG
TCGCTTGCGG
TGGTCCCGCC
TGGCGGCCGA
TGGATGGCCT
GCCCGCGTTG
GACAGCTTCA
GGACCGCTGA
CGGGTTGGCA
CGTTGCGTCG
ATTAAAGGCT
ATCAAAGGAT
GCAAACAAAA
AGCTACCAAC
CCAAATACTG
CTCTGTAGCA
CTGCTGCCAG
TAGTTACCGG
ACAGCCCAGC
GTGAGCTATG
TATCCGGTAA
AGGGGGAAAC
GACTTGAGCG
AAAAACGCCA
TTTTGCTCAC
GTATTACCGC
GAGCGCAGCG
TTTTCTCCTT
CAGTACAATC
ATCGCTACGT
CTGACGCGCC
GCTGTGACCG
ACCGAAACGC
GCTATACGAA
AGTTCAACCT
CATGCATAAC
GACTCACTAT
ATTTTGTTTA
TAAAACGACG
GTCGAGCTCG
GAACGCGTCT
GTTGGCTGCT
CTAAACGGGT
GTAGCCCGCC
TTTCTAAATA
AAATGCTTCA
ACEMBL MultiColi
ACAATGCGCT
ATAGGCTTGG
TTCCGACAGC
TGATGCAATT
GGCCGCCGCC
CGCGATCATG
TCGTGGCCGG
GCCGACATCA
CGCTTGTTTC
TGGGCGCCAT
GGCCTCAACC
AGAGCGCCGA
GGTGGGCGCG
ATCATGCAAC
CGAGGACCGC
TATTCGGAAT
ACCAAACGTT
CGCGCTGGGC
TCCCCATTAT
CAGGCCATGC
AGGATCGCTC
TCGTCACGGC
TGGATTGTAG
CGGTGCATGG
CCTTTTGGAG
CTTCTTGAGA
AAACCACCGC
TCTTTTTCCG
TCCTTCTAGT
CCGCCTACAT
TGGCGATAAG
ATAAGGCGCA
TTGGAGCGAA
AGAAAGCGCC
GCGGCAGGGT
GCCTGGTATC
TCGATTTTTG
GCAACGCGGC
ATGTTCTTTC
CTTTGAGTGA
AGTCAGTGAG
ACGCATCTGT
TGCTCTGATG
GACTGGGTCA
CTGACGGGCT
TCTCCGGGAG
GCGAGGCAGG
GTTATGGTAC
GTTGATAGTA
TATAACGGTC
AGGGGAATTG
ACTTTAAGAA
GCCAGTGAAT
ATATCGGTAC
CGAGAGATCC
GCCACCGCTG
CTTGAGGGGT
TAATGAGCGG
CATTCAAATA
ATAATATTGA
CATCGTCATC
TTATGCCGGT
ATCGCCAGTC
TCTATGCGCA
CAGTCCTGCT
GCGACCACAC
CATCACCGGC
CCGATGGGGA
GGCGTGGGTA
CTCCTTACAT
TACTACTGGG
CCCATGCCCT
GGGCATGACT
TCGTAGGACA
TTTCGCTGGA
CTTGCACGCC
TCGGCGAGAA
TACGTCTTGC
GATTCTTCTC
TGTCCAGGCA
GCGGCTCTTA
GATTTATGCC
GCGCCGCCCT
AGCCGGGCCA
CCTTTTTTTT
TCCTTTTTTT
TACCAGCGGT
AAGGTAACTG
GTAGCCGTAG
ACCTCGCTCT
TCGTGTCTTA
GCGGTCGGGC
CGACCTACAC
ACGCTTCCCG
CGGAACAGGA
TTTATAGTCC
TGATGCTCGT
CTTTTTACGG
CTGCGTTATC
GCTGATACCG
CGAGGAAGCG
GCGGTATTTC
CCGCATAGTT
TGGCTGCGCC
TGTCTGCTCC
CTGCATGTGT
GGGAATTCCA
CGCGGCCGCG
CTTCGTTAAT
CTAAGGTAGC
TGAGCGGATA
GGAGATATAC
TCCCCGGGAA
CAGCGGATAA
GGCTGCTAAC
AGCAATAACT
TTTTTGGTTT
GCTTTTTTTT
TGTATCCGCT
AAAAGGAAGA
CTCGGCACCG
ACTGCCGGGC
ACTATGGCGT
CCCGTTCTCG
CGCTTCGCTA
CCGTCCTGTG
GCCACAGGTG
AGATCGGGCT
TGGTGGCAGG
GCACCATTCC
CTGCTTCCTA
TGAGAGCCTT
ATCGTCGCCG
GGTGCCGGCA
GCGCGACGAT
CTCGCTCAAG
GCAGGCCATT
TGGCGTTCGC
GCTTCCGGCG
GGTAGATGAC
CCAGCCTAAC
GCCTCGGCGA
ATACCTTGTC
CCTCGACCTG
TGGACGGACC
CTGCGCGTAA
GGTTTGTTTG
GCTTCAGCAG
TTAGGCCACC
GCTAATCCTG
CCGGGTTGGA
TGAACGGGGG
CGAACTGAGA
AAGGGAGAAA
GAGCGCACGA
TGTCGGGTTT
CAGGGGGGCG
TTCCTGGCCT
CCCTGATTCT
CTCGCCGCAG
GAAGAGCGCC
ACACCGCAAT
AAGCCAGTAT
CCGACACCCG
CGGCATCCGC
CAGAGGTTTT
GATAACTTCG
TAGAGGATCT
ACAGATGTAG
GACCTAGGTA
ACAATTCCCC
ATATGAGGCC
GCTTCGCCAG
CAATTTCACA
AAAGCCCGAA
AGCATAACCC
AAACCCATCT
AATTCCCCTA
CATGAGACAA
GT
TCACCCTGGA
CTCTTGCGGG
GCTGCTAGCG
GAGCACTGTC
CTTGGAGCCA
GATTCTCTAC
CGGTTGCTGG
CGCCACTTCG
CCCCGTGGCC
TTGCGGCGGC
ATGCAGGAGT
CAACCCAGTC
CACTTATGAC
GCGCTCTGGG
GATCGGCCTG
CCTTCGTCAC
ATCGCCGGCA
GACGCGAGGC
GCATCGGGAT
GACCATCAGG
TTCGATCATT
GCACATGGAA
TGCCTCCCCG
AACCGATACA
GGTAGAAAAG
TCTGCTGCTT
CCGGATCAAG
AGCGCAGATA
ACTTCAAGAA
TTACCAGTGG
CTCAAGACGA
GTTCGTGCAC
TACCTACAGC
GGCGGACAGG
GGGAGCTTCC
CGCCACCTCT
GAGCCTATGG
TTTGCTGGCC
GTGGATAACC
CCGAACGACC
TGATGCGGTA
GGTGCACTCT
ACACTCCGCT
CCAACACCCG
TTACAGACAA
CACCGTCATC
TATAATGTAT
GTTGATCAGC
GTGTTGGCAC
TCGATAATAC
TCTAGAAATA
TCGGATCCTG
GGTTTTCCCA
TCCGGATCGC
AGGAAGCTGA
CTTGGGGCCT
AATTGGACTA
TTTGTTTATT
TAACCCTGAT
June 2011
43
F.2.3. pDC
1
51
101
151
201
251
301
351
401
451
501
551
601
651
701
751
801
851
901
951
1001
1051
1101
1151
1201
1251
1301
1351
1401
1451
1501
1551
1601
1651
1701
1751
1801
1851
1901
1951
2001
2051
ATCAACGTCT
GAGAAAGAGG
TAGGGGAATT
AACTTTAAGA
GGCCAGTGAA
GATATCGGTA
TCGAGAGATC
TGCCACCGCT
TCTTGAGGGG
ATAATGTATG
TTGATCAGCA
ACGTACTAAG
TAAACCCTCA
CTCATGTTTC
AATCAGCAAC
AGAATATATA
AAGCCAGGGA
TTTGAGTGAC
CCGCAAGCAC
AAGCCAGTCC
GCAGAATAAA
AGCAAACTGG
TAGTCAATAA
CCCTGCCCTG
TTCATCCGCT
GCACCAATAA
AGTACTGTTG
ACGGCATGAT
CGTATAATAT
TGGCCACGTT
ACGAAAAACA
ACCGTAACAC
CGTCGTGGTA
AAAACGGTGT
TTTCATTGCC
TGTGAATAAA
AAAAAGGCCG
AACTGACTGA
CAACGGTGGT
CCTGAAAATC
ATTATGGTGA
GCCAAAAGTT
ACEMBL MultiColi
CATTTTCGCC
TAATGAAATG
GTGAGCGGAT
AGGAGATATA
TTCCCCGGGA
CCAGCGGATA
CGGCTGCTAA
GAGCAATAAC
TTTTTTGGTT
CTATACGAAG
GTTCAACCTG
CTCTCATGTT
TGGCTAACGT
ACGTACTAAG
TTAAATAGCC
AGGCTTTTAA
TGTAACGCAC
ACAGGAACAC
TCAGGGCGCA
GCAGAAACGG
TGATCATATC
CCTCAGGCAT
ACCGGTAAAC
AACCGACGAC
TATTATCACT
CTGCCTTAAA
TAATTCATTA
GAACCTGAAT
TTGCCCATGG
TAAATCAAAA
TATTCTCAAT
GCCACATCTT
TTCACTCCAG
AACAAGGGTG
ATACGGAATT
GGCCGGATAA
TAATATCCAG
AATGCCTCAA
ATATCCAGTG
TCGATAACTC
AAGTTGGACC
GGCCCAG
AAAAGTTGGC
GCACCTAGGT
AACAATTCCC
CATATGAGGC
AGCTTCGCCA
ACAATTTCAC
CAAAGCCCGA
TAGCATAACC
TAAACCCATG
TTATGGTACC
TTGATAGTAC
TAACGTACTA
ACTAAGCTCT
CTCTCATGTT
TCTAAGGTTT
AGCTTTTAAG
TGAGAAGCCC
TTAACGGCTG
AGGGCTGCTA
TGCTGACCCC
GTCAATTATT
TTGAGAAGCA
CAGCAATAGA
CGGGTCGAAT
TATTCAGGCG
AAAATTACGC
AGCATTCTGC
CGCCAGCGGC
TGAAAACGGG
CTGGTGAAAC
AAACCCTTTA
GCGAATATAT
AGCGATGAAA
AACACTATCC
CCGGATGAGC
AACTTGTGCT
CTGAACGGTC
AATGTTCTTT
ATTTTTTTCT
AAAAAATACG
CTCTTACGTG
CCAGATCTAT
ATCGATAATA
CTCTAGAAAT
CTCGGATCCT
GGGTTTTCCC
ATCCGGATCG
AAGGAAGCTG
CCTTGGGGCC
TGCCTGGCAG
GCGGCCGCGT
GTACTAAGCT
AGCTCTCATG
CATGGCTAAC
TGAACAATAA
TAAGTTTTAT
GTTTAACGGT
TTAGAGCCTC
ACAGAATTAG
AAGGAAGCGG
GGATGAATGT
ACCTCCACGG
CACGGTCACA
CATAAGCGGC
TTGCTTTCGA
TAGCAACCAG
CCCGCCCTGC
CGACATGGAA
ATCAGCACCT
GGCGAAGAAG
TCACCCAGGG
GGGAAATAGG
GTGTAGAAAC
ACGTTTCAGT
CATATCACCA
ATTCATCAGG
TATTTTTCTT
TGGTTATAGG
ACGATGCCAT
CCATTTTAGC
CCCGGTAGTG
CCGATCAACG
GTCGGGTGCG
CGACTCACTA
AATTTTGTTT
GTAAAACGAC
AGTCGAGCTC
CGAACGCGTC
AGTTGGCTGC
TCTAAACGGG
ATAACTTCGT
AGAGGATCTG
CTCATGTTTC
TTTAACGAAC
GTACTAAGCT
AATTAATATA
AAGAAAAAAA
TGTGGACAAC
TCAAAGCAAT
CTTCACGCTG
AACACGTAGA
CAGCTGGGAG
GGAGAGCCTG
CTGCTTCCGG
TATTTAACGA
ATTTCTGCCA
GCGTTTAAGG
CACTCATCGC
GCCATCACAA
TGTCGCCTTG
TTGTCCATAT
ATTGGCTGAG
CCAGGTTTTC
TGCCGGAAAT
TTGCTCATGG
GCTCACCGTC
CGGGCAAGAA
TACGGTCTTT
TACATTGAGC
TGGGATATAT
TTCCTTAGCT
ATCTTATTTC
TCTCATTTTC
June 2011
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F.2.4. pDK
1
51
101
151
201
251
301
351
401
451
501
551
601
651
701
751
801
851
901
951
1001
1051
1101
1151
1201
1251
1301
1351
1401
1451
1501
1551
1601
1651
1701
1751
1801
1851
1901
1951
2001
2051
CTATGTCGGG
GGCTTTACAC
GATAACAATT
GAAATAATTT
ATCCTGTAAA
TTCCCAGTCG
GATCGCGAAC
AGATAACTTC
TCTCATGTTT
GTTTAACGAA
CGTACTAAGC
AAATTAATAT
TAAGAAAAAA
TTGTGGACAA
CTCAAAGCAA
GCTTCACGCT
GAACACGTAG
TCAGCTACTG
CAGGTAGCTT
ATGGACAGCA
TTGGGAAGCC
TGATGGCGCA
TCGCATGATT
TGGAGAGGCT
GATGCCGCCG
CAAGACCGAC
GGCTATCGTG
GTTGTCACTG
GCAGGATCTC
TGGCTGATGC
TTCGACCACC
AGCCGGTCTT
CGCCAGCCGA
GATCTCGTCG
AAATGGCCGC
ACCGCTATCA
GGCGGCGAAT
CGATTCGCAG
CGGGACTCTG
CACGAGATTT
ATCGTTTTCC
GCTGGAGTTC
ACEMBL MultiColi
TGCGGAGAAA
TTTATGCTTC
TCACACAGGA
TGTTTAACTT
ACGACGGCCA
AGCTCGATAT
GCGTCTCGAG
GTATAATGTA
CACGTACTAA
CTAAACCCTC
TCTCATGTTT
AAATCAGCAA
AAGAATATAT
CAAGCCAGGG
TTTTCAGTGA
GCCGCAAGCA
AAAGCCAGTC
GGCTATCTGG
GCAGTGGGCT
AGCGAACCGG
CTGCAAAGTA
GGGGATCAAG
GAACAAGATG
ATTCGGCTAT
TGTTCCGGCT
CTGTCCGGTG
GCTGGCCACG
AAGCGGGAAG
CTGTCATCTC
AATGCGGCGG
AAGCGAAACA
GTCGATCAGG
ACTGTTCGCC
TGACACATGG
TTTTCTGGAT
GGACATAGCG
GGGCTGACCG
CGCATCGCCT
GGGTTCGAAA
CGATTCCACC
GGGACGCCGG
TTCGCCCACC
GAGGTAATGA
CGGCTCGTAT
AACAGCTATG
TAAGAAGGAG
GTGAATTCCC
CGGTACCAGC
ACTAGTTCCG
TGCTATACGA
GCTCTCATGT
ATGGCTAACG
CACGTACTAA
CTTAAATAGC
AAGGCTTTTA
ATGTAACGCA
CACAGGAACA
CTCAGGGCGC
CGCAGAAACG
ACAAGGGAAA
TACATGGCGA
AATTGCCAGC
AACTGGATGG
ATCTGATCAA
GATTGCACGC
GACTGGGCAC
GTCAGCGCAG
CCCTGAATGA
ACGGGCGTTC
GGACTGGCTG
ACCTTGCTCC
CTGCATACGC
TCGCATCGAG
ATGATCTGGA
AGGCTCAAGG
CGATGCCTGC
TCATCGACTG
TTGGCTACCC
CTTCCTCGTG
TCTATCGCCT
TGACCGACCA
GCCGCCTTCT
CTGGATGATC
CCGGGAT
AATGGCACCT
GTTGTGTGGA
ACCATGATTA
ATATACATAT
CGGGAAGCTT
GGATAACAAT
TTTAAACCCA
AGTTATGGTA
TTAACGTACT
TACTAAGCTC
GCTCTCATGT
CTCTAAGGTT
AAGCTTTTAA
CTGAGAAGCC
CTTAACGGCT
AAGGGCTGCT
GTGCTGACCC
ACGCAAGCGC
TAGCTAGACT
TGGGGCGCCC
CTTTCTTGCC
GAGACAGGAT
AGGTTCTCCG
AACAGACAAT
GGGCGCCCGG
ACTGCAGGAC
CTTGCGCAGC
CTATTGGGCG
TGCCGAGAAA
TTGATCCGGC
CGAGCACGTA
CGAAGAGCAT
CGCGCATGCC
TTGCCGAATA
TGGCCGGCTG
GTGATATTGC
CTTTACGGTA
TCTTGACGAG
AGCGACGCCC
ATGAAAGGTT
CTCCAGCGCG
AGGTATCGAT
ATTGTGAGCG
CGAATTTCTA
GAGGCCTCGG
CGCCAGGGTT
TTCACATCCG
TGTGCCTGGC
CGTACTAAGC
AAGCTCTCAT
TCATGGCTAA
TTGAACAATA
TTAAGTTTTA
GGTTTAACGG
CTTAGAGCCT
GACAGAATTA
AAAGGAAGCG
CGGATGAATG
AAAGAGAAAG
GGGCGGTTTT
TCTGGTAAGG
GCCAAGGATC
GAGGATCGTT
GCCGCTTGGG
CGGCTGCTCT
TTCTTTTTGT
GAGGCAGCGC
TGTGCTCGAC
AAGTGCCGGG
GTATCCATCA
TACCTGCCCA
CTCGGATGGA
CAGGGGCTCG
CGACGGCGAG
TCATGGTGGA
GGTGTGGCGG
TGAAGAGCTT
TCGCCGCTCC
TTCTTCTGAG
AACCTGCCAT
GGGCTTCGGA
GGGATCTCAT
June 2011
45
F.2.5. pDS
1
51
101
151
201
251
301
351
401
451
501
551
601
651
701
751
801
851
901
951
1001
1051
1101
1151
1201
1251
1301
1351
1401
1451
1501
1551
1601
1651
1701
1751
1801
1851
1901
1951
2001
CTATGTCGGG
GGCTTTACAC
GATAACAATT
GAAATAATTT
ATCCTGTAAA
TTCCCAGTCG
GATCGCGAAC
AGATAACTTC
TCTCATGTTT
GTTTAACGAA
CGTACTAAGC
AAATTAATAT
TAAGAAAAAA
TTGTGGACAA
CTCAAAGCAA
AGCTTCACGC
GGAACACGTA
GTCAGCTGGG
GGGGAGAGCC
CACTGCTTCC
ACTGGTCCAG
GTTTTCATGG
GGCATCCAAG
GGAGCAGCAA
AACATCATGA
GGTAGTTGGC
ATTTGTACGG
GATTTGCTGG
TTTGATCAAC
TTCTCCGCGC
TGGCGTTATC
TGACATTCTT
CTATCTTGCT
GCGGCGGAGG
GCTAAATGAA
ATGAGCGAAA
ACCGGCAAAA
CCTGCCGGCC
TTGGACAAGA
TTTGTCCACT
TCTAACAATT
ACEMBL MultiColi
TGCGGAGAAA
TTTATGCTTC
TCACACAGGA
TGTTTAACTT
ACGACGGCCA
AGCTCGATAT
GCGTCTCGAG
GTATAATGTA
CACGTACTAA
CTAAACCCTC
TCTCATGTTT
AAATCAGCAA
AAGAATATAT
CAAGCCAGGG
TTTTGAGTGA
TGCCGCAAGC
GAAAGCCAGT
AGGCAGAATA
TGAGCAAACT
GGTAGTCAAT
AACCTTGACC
CTTGTTATGA
CAGCAAGCGC
CGATGTTACG
GGGAAGCGGT
GTCATCGAGC
CTCCGCAGTG
TTACGGTGAC
GACCTTTTGG
TGTAGAAGTC
CAGCTAAGCG
GCAGGTATCT
GACAAAAGCA
AACTCTTTGA
ACCTTAACGC
TGTAGTGCTT
TCGCGCCGAA
CAGTATCAGC
AGAAGATCGC
ACGTGAAAGG
CGTTCAAGCC
GAGGTAATGA
CGGCTCGTAT
AACAGCTATG
TAAGAAGGAG
GTGAATTCCC
CGGTACCAGC
ACTAGTTCCG
TGCTATACGA
GCTCTCATGT
ATGGCTAACG
CACGTACTAA
CTTAAATAGC
AAGGCTTTTA
ATGTAACGCA
CACAGGAACA
ACTCAGGGCG
CCGCAGAAAC
AATGATCATA
GGCCTCAGGC
AAACCGGTAA
GAACGCAGCG
CTGTTTTTTT
GTTACGCCGT
CAGCAGGGCA
GATCGCCGAA
GCCATCTCGA
GATGGCGGCC
CGTAAGGCTT
AAACTTCGGC
ACCATTGTTG
CGAACTGCAA
TCGAGCCAGC
AGAGAACATA
TCCGGTTCCT
TATGGAACTC
ACGTTGTCCC
GGATGTCGCT
CCGTCATACT
TTGGCCTCGC
CGAGATCACC
GACGGAT
AATGGCACCT
GTTGTGTGGA
ACCATGATTA
ATATACATAT
CGGGAAGCTT
GGATAACAAT
TTTAAACCCA
AGTTATGGTA
TTAACGTACT
TACTAAGCTC
GCTCTCATGT
CTCTAAGGTT
AAGCTTTTAA
CTGAGAAGCC
CTTAACGGCT
CAAGGGCTGC
GGTGCTGACC
TCGTCAATTA
ATTTGAGAAG
GTAGCGTATG
GTGGTAACGG
GGGGTACAGT
GGGTCGATGT
GTCGCCCTAA
GTATCGACTC
ACCGACGTTG
TGAAGCCACA
GATGAAACAA
TTCCCCTGGA
TGCACGACGA
TTTGGAGAAT
CACGATCGAC
GCGTTGCCTT
GAACAGGATC
GCCGCCCGAC
GCATTTGGTA
GCCGACTGGG
TGAAGCTAGA
GCGCAGATCA
AAGGTAGTCG
AGGTATCGAT
ATTGTGAGCG
CGAATTTCTA
GAGGCCTCGG
CGCCAGGGTT
TTCACATCCG
TGTGCCTGGC
CGTACTAAGC
AAGCTCTCAT
TCATGGCTAA
TTGAACAATA
TTAAGTTTTA
GGTTTAACGG
CTTAGAGCCT
GACATAATTC
TAAAGGAAGC
CCGGATGAAT
TTACCTCCAC
CACACGGTCA
CGCTCACGCA
CGCAGTGGCG
CTATGCCTCG
TTGATGTTAT
AACAAAGTTA
AACTATCAGA
CTGGCCGTAC
CAGTGATATT
CGCGGCGAGC
GAGAGCGAGA
CATCATTCCG
GGCAGCGCAA
ATTGATCTGG
GGTAGGTCCA
TATTTGAGGC
TGGGCTGGCG
CAGCGCAGTA
CAATGGAGCG
CAGGCTTATC
GTTGGAAGAA
GCAAATAATG
June 2011
46
F.3. ACEMBL plasmid maps
Acceptor vectors pACE1 (pACE) and pACE2, containing a T7 promoter and terminator, are
shown. Donor vectors pDK, pDS and pDC contain conditional origins of replication. pDS
and pDK have a lac promoter. pDC has a T7 promoter. Resistance markers are shown in
gray, origins of replication in yellow. LoxP imperfect inverted repeat sequences are shown
as spheres. Homing endonuclease sites and corresponding BstXI sites are boxed. The
restriction enzyme sites in the multiple integration element (MIE) are indicated. All MIEs
ACEMBL MultiColi
June 2011
47
have the same DNA sequence between ClaI and PmeI. Differences in unique restriction
site composition stem from differences in the plasmid backbone sequences.
ACEMBL MultiColi
June 2011
48
F.4. Analytical restriction of ACEMBL vectors
All ACEMBL vectors were analyzed by BamHI restriction digestion. The undigested and
digested ACEMBL vectors are shown below:
1
2
3
4
5
M
A
B
C
D
E
Restriction mapping of ACEMBL vectors. Both undigested as well as BamHI-digested Acceptor
and Donor vectors are. All restriction reactions yield the expected sizes. Lane 1-5 show uncut
pACE1, pACE2, pDC, pDK, and pDS vectors; lane M shows λ StyI marker; lane A-E show BamHIdigested pACE1, pACE2, pDC, pDK, and pDS vectors.
Additionally, the MultiColi vectors yield characteristic restriction digest patterns when
plasmid DNA is restricted with HindIII and HinfI.
Vector
Length [bp]
Restriction
Fragment(s) [bp]
pACE1
2652
HindIII
2652
HinfI
1690, 491, 396, 75
HindIII
2982
HinfI
772, 618, 491, 396, 256, 220, 154, 75
HindIII
1517, 550
HinfI
1275, 792
HindIII
1681, 396
HinfI
1796, 134, 58, 52, 37
HindIII
1631, 396
HinfI
1814, 213
pACE2
pDC
pDK
pDS
2982
2067
2077
2027
ACEMBL MultiColi
June 2011
49
G. References
1.
Alberts B (1998). The cell as a collection of protein machines: preparing the next generation
of molecular biologists. Cell 92: 291-294.
2.
Ausubel F, Brent R, et al. (eds., 1994). Current Protocols in Molecular Biology. John Wiley &
Sons, New York, electronic version DOI 10.1002/0471142727
3.
Bhattacharya A (2009). Protein structure: Structures of desire. Nature 459: 24-27.
4.
Bieniossek C, Nie Y, Frey D, Olieric N, Schaffitzel C, Collinson I, Romier C, Berger P,
Richmond TJ, Steinmetz MO, Berger I (2009). Automated unrestricted multigene
recombineering for multiprotein complex production. Nature Methods 6: 447-450.
5.
Charbonnier S, Gallego O, Gavin AC (2008). The social network of a cell: Recent advances in
interactome mapping. Biotechnology Annual Review 14: 1-28.
6.
Chemler JA and Koffas MAG (2008). Metabolic engineering for plant natural product
biosynthesis in microbes. Current Opinion in Biotechnology 19: 597-605.
7.
Chou CP (2007). Engineering cell physiology to enhance recombinant protein production in
Escherichia coli. Applied Microbiology and Biotechnology 76: 521-532.
8.
Estrozi LF, Boehringer D, Shan SO, Ban N, Schaffitzel C (2011). Cryo-EM structure of the E.
coli translating ribosome in complex with SRP and its receptor. Nature Structural and
Molecular Biology 18: 88-90.
9.
Gaiser F, Tan S, and Richmond TJ (2000). Novel dimerization fold of RAP30/RAP74 in human
TFIIF at 1.7 Å resolution. Journal of Molecular Biology 302: 1119-1127.
10. Inoue H, Nojima H and Okayama H (1990). High efficiency transformation of Escherichia coli
with plasmids. Gene 96: 23-28.
11. Lee SK, Chou H, Ham TS, Lee TS, and Keasling JD (2008). Metabolic engineering of
microorganisms for biofuels production: from bugs to synthetic biology to fuels. Current
Opinion in Biotechnology 19: 556-563.
12. Li M and Elledge SJ (2007). Harnessing homologous recombination in vitro to generate
recombinant DNA via SLIC. Nature Methods 4: 251-256.
13. Nie Y, Viola C, Bieniossek C, Trowitzsch S, Vijay-Achandran LS, Chaillet M, Garzoni F, Berger
I (2009). Getting a grip on complexes. Current Genomics 10: 558-72.
14. Robinson CV, Sali A, and Baumeister W (2007). The molecular sociology of the cell. Nature
450: 973-982.
15. Sambrook J and Russell DW (2000). Molecular Cloning, A Laboratory Manual, 3rd edition.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, available online at
http://cshprotocols.cshlp.org
16. Tan S, Kern RC, and Selleck W (2005). The pST44 polycistronic expression system for
producing protein complexes in Escherichia coli. Protein Expression and Purification 40: 385395.
17. Van Hijum SA, Medema MH, and Kuipers OP (2009). Mechanisms and evolution of control
logic in prokaryotic transcriptional regulation. Microbiology and Molecular Biology Reviews
73: 481-509.
Please consider quoting the boxed article as original reference when publishing
results obtained with the MultiColio kit.
ACEMBL MultiColi
June 2011

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