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Keywords: recombination; cloning; clonase;
one-pot reaction
A)
B)
R
Method summary: We developed efficient,
cost and time saving protocols that
enable the isolation of Gateway entry and
expression clones using a single consolidated reaction capable of simultaneously
performing BP and LR recombination steps.
Vol. 55 | No. 5 | 2013
attB1
Gene
Gene
Expression Clone
1
attP1
attB2
Expression
Clone
Gene
Expression Clone
6
attP2
Donor
Vector
+
R
(Amp )
ccdB
R
(Kan )
Gene
Gene
Expression Clone
2
Entry Clone
Gene
Expression Clone
3
Gene
Expression Clone
5
Gene
Expression Clone
4
LR Clonase®
(Int + IHF + Xis)
BP Clonase®
(Int + IHF)
attL1
attR1
attL2
Entry
Clone
E
P
R
IN
T
The Gateway recombination system is
characterized by its ability to transfer DNA
sequences back and forth between an intermediate clone (the entry clone) and a variety
of destination vectors. However, a number
of applications do not need to exploit the
advantages offered by the entry clone. Here
we report reaction conditions for cloning
DNA fragments into destination vectors
in a single step reaction, thus reducing the
cost and overall time needed to obtain an
expression clone from three days to one.
IO
N
S
BioTechniques 55:265-268 (November 2013)
doi 10.2144/000114101
thereby reducing the time needed to obtain
a final expression clone from three days to
one.
The rationale for our approach stems
from the fact that the main difference
between the BP and LR reactions is the
presence of the lambda excisionase (Xis)
in the LR Clonase blend, which drives the
recombination dynamics toward phage
excision (3). Therefore, there should be an
excisionase level where both reactions occur
at similar rate.
To facilitate the fine tuning of the BP/LR
combined reaction, we employed a DNA
fragment harboring the lacZα reporter gene.
The linear double-stranded DNA fragment
was used as delivered by a gene synthesis
provider, without precloning or prior PCR
amplification. Reactions were set up using
different ratios of BP and LR Clonase
in the presence of the reporter sequence
as well as donor and destination vectors
(see Supplementary Material). Successful
recombination events into both vectors were
represented as blue colonies selected on
X-gal agar plates supplemented with the
corresponding antibiotics.
IS
Life Technologies, Carlsbad, CA
The Gateway cloning system exploits
the site-specific recombination system
utilized by bacteriophage lambda to shuttle
sequences between plasmids bearing
flanking compatible recombination
attachment (att) sites (1). Once captured
as an entry clone, a DNA fragment can be
recombined into a variety of destination
vectors resulting in expression clones geared
to specific applications (Figure 1A). The
recombination reactions are driven by two
enzyme blends known by their commercial
names: BP Clonase and LR Clonase (Figure
1B). Further details on the Gateway system
can be found in an earlier review (2).
One of the drawbacks of the system is
that, starting from a linear PCR fragment,
it takes two bacterial transformation steps
(or three days) to obtain the final expression
clone. For those applications that do not
require the isolation of an entry clone,
the workflow adds two days to the overall
cloning timeframe compared with other
cloning approaches.
Here we describe a new approach
combining the two Gateway steps, BP and
LR recombination, into a single reaction,
M
Xiquan Liang, Lansha Peng,
Chang-Ho Baek, and Federico
Katzen
O
N
Single step BP/LR combined Gateway reactions
(KanR)
+
ccdB
attR2
Destination
Vector
(AmpR)
Figure 1. The Gateway recombination system. (A) A DNA fragment containing the gene of interest is recombined into a donor vector
using the BP reaction (single-headed arrow) resulting in an entry clone. This clone can be used in an LR reaction (double-headed arrows)
to transfer the fragment into multiple destination vectors, generating expression clones. The gene of interest in the expression clones may
be transferred back by a BP reaction to generate an entry clone (double-headed arrows). (B) The BP Clonase contains the phage lambda
integrase (int) and the E. coli integration host factor (IHF) and transfers DNA fragments flanked by attB sequences into a donor vector,
which has the counterselectable marker ccdB flanked by attP sequences. The LR Clonase blend has the same enzyme components
as the BP Clonase blend, plus the lambda excisionase (Xis), promoting the opposite reaction where a fragment is transferred from
an entry clone into a destination vector generating the expression clone. AmpR, ampicillin resistance; KanR, kanamycin resistance.
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B)
AmpR colonies - Expression clones (LacZ-α)
100
80
10000
80
60
1000
40
100
20
10
1
10000
1000
60
100
40
10
20
0
LR Clonase® (%)
0
0*
0
25
50
75
100
0
LR Clonase® (%)
0
0*
0
25
50
75
100
BP Clonase® (%)
0
100*
100
75
50
25
0
BP Clonase® (%)
0
100*
100
75
50
25
0
CE (% Blue colonies)
1
CE (% Blue colonies)
CFU/plate
D)
KanR colonies - Entry clones (GFP)
1000000
80
100000
10000
60
1000
40
100
20
CFU/plate
AmpR colonies - Expression clones (GFP)
100
CE (% fluorescent colonies)
100
CFU/plate
CE (%pENTR+ clones)
C)
10
10000
80
1000
60
100
40
10
20
0
1
1
0
LR Clonase® (%)
0
0
75
100
LR Clonase® (%)
0
0
75
100
BP Clonase® (%)
0
100*
25
0
BP Clonase® (%)
0
100*
25
0
CE (% pENTR+ clones)
CE (% fluorescent colonies)
CFU/plate pENTR
CFU/plate
100000
CE (% blue colonies)
100
CFU/plate
KanR colonies - Entry clones (LacZ-α)
CFU/plate
CE (% blue colonies)
A)
CFU/plate
Figure 2. Efficiencies of consolidated BP and LR reactions. Reactions were set up using 100 ng of a linear synthesized fragment (A and B) or a PCR fragment (C and
D), pDONR-221 (donor vector), and pEXP1-Dest (destination vector). Enzymes were added in the indicated proportion. Total reaction volume was kept at 10 µL.
(A and C) Cloning efficiency (CE) and colony count (CFU/plate) on LB kanamycin X-gal agar plates. (B and D) CE and CFU/plate on LB ampicillin X-gal agar plates.
Asterisks represent reactions where pEXP1-Dest DNA was purposely omitted. For further details see Supplementary Material. The number of green fluorescent colonies
in (D) was determined by placing the plates on top of a UV transilluminator. Entry clones containing the PTAC-GFP fragment in (C) exhibited very little fluorescence and
were virtually indistinguishable from truly non-fluorescent colonies. The presence of the insert in this case was validated by colony-PCR assays using M13-forward
and reverse oligonucleotides. Clones with the expected insert are indicated as “pENTR+ clones.” AmpR, ampicillin resistance; KanR, kanamycin resistance.
Vol. 55 | No. 5 | 2013
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Benchmark
The results showed that at elevated
LR/BP ratios the product is enriched in
the expression clone, whereas opposite
proportions produce an enrichment of
the entry clone (Figure 2A and 2B). The
cloning efficiencies depicted in Figure
2A and 2B are based exclusively on the
percentage of blue colonies, and therefore
underestimate the real recombination
efficiency. For example, DNA sequencing
of four random kanamycin resistant white
colonies revealed that all four contain the
right insert with internal single nucleotide
mutations, reflecting errors during DNA
synthesis. On the other hand, the sequence
of 10 random ampicillin resistant white
colonies showed that half contained
similar errors as above. Equivalent results
were obtained when a PCR fragment
encoding the GFP gene preceded by the
tac promoter was used instead of the lacZα
gene (Figure 2C and D), suggesting that
the method is generally applicable.
Overall our results showed that BP
and LR recombination can proceed
at high efficiencies in a single tube
without the need for subsequent steps.
If the exclusive goal is to obtain an
expression clone, the use of LR Clonase
alone is the preferred option, as it can
perform both recombination events,
producing thousands of expression
clones. Under these conditions, when
the excisionase protein is present, BP
Vol. 55 | No. 5 | 2013
recombination operates at a lower rate
and, as expected, a reduced number of
entry clones are obtained. This number,
although sufficiently large for most cases,
may fluctuate depending on the nature
and quality of the insert and vectors.
Methods to further increase the chances
of simultaneous entry clone selection
include (i) using electroporation rather
than chemical transformation protocols,
or (ii) raising the BP:LR Clonase ratio to
1:3.
The strategy presented here could
be expanded to cover the assembly of
multiple fragments into a single vector
as proposed previously (4). However, the
isolation of individual entry clones should
be subjected to screening as all of them
will likely provide the same antibiotic
resistance.
Finally, our approach can also be
applied to transfer an insert from an
expression clone to a second destination
vector in a single step. In this case a
linearization of the expression clone will
be necessary to reduce the background,
unless the antibiotic resistance markers of
the two episomes are different.
Author contributions
XL, CHB, and LP performed the
experiments. FK conceived the idea and
wrote the manuscript.
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Competing interests
The authors hold financial interests in Life
Technologies.
References
1. Hartley, J.L., G.F. Temple, and M.A.
Brasch. 2000. DNA cloning using in vitro
site-specific recombination. Genome Res.
10:1788-1795.
2. Katzen, F. 2007. Gateway recombinational
cloning: a biological operating system. Expert
Opin Drug Discov. 2:571-589.
3. Landy, A. 1989. Dynamic, structural, and
regulatory aspects of lambda site-specific
recombination. Annu. Rev. Biochem. 58:913949.
4. Cheo, D.L., S.A. Titus, D.R. Byrd, J.L.
Hartley, G.F. Temple, and M.A. Brasch.
2004. Concerted assembly and cloning of
multiple DNA segments using in vitro site-specific recombination: functional analysis of
multi-segment expression clones. Genome
Res. 14:2111-2120.
Received 29 August 2013; accepted 16 October
2013.
Address correspondence to Federico Katzen,
Life Technologies, Carlsbad, CA. E-mail:
federico.katzen@lifetech.com
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