Appl Microbiol Biotechnol (2012) 93:18531863

DOI 10.1007/s00253-012-3920-8

MINI-REVIEW

Available methods for assembling expression cassettes

for synthetic biology
Tianwen Wang & Xingyuan Ma & Hu Zhu & Aitao Li &
Guocheng Du & Jian Chen

Received: 8 December 2011 / Revised: 19 January 2012 / Accepted: 20 January 2012 / Published online: 7 February 2012
# Springer-Verlag 2012

Abstract Studies in the structural biology of the multicomponent protein complex, metabolic engineering, and synthetic
biology frequently rely on the efficient over-expression of
these subunits or enzymes in the same cell. As a first step,
constructing the multiple expression cassettes will be a complicated and time-consuming job if the classic and conventional digestion and ligation based cloning method is used.
Some more efficient methods have been developed, including
(1) the employment of a multiple compatible plasmid expression system, (2) the rare-cutter-based design of vectors, (3) in
vitro recombination (sequence and ligation independent cloning, the isothermally enzymatic assembly of DNA molecules
in a single reaction), and (4) in vivo recombination using
recombination-efficient yeast (in vivo assembly of overlapping fragments, reiterative recombination for the chromosome
integration of foreign expression cassettes). In this review, we
systematically introduce these available methods.
Keywords Synthetic biology . Simultaneous expression .
Pathway construction . Yeast recombination . Cre-loxP sitespecific recombination . Acembl system
T. Wang : G. Du : J. Chen
State Key Laboratory of Food Science and Technology,
Jiangnan University,
Wuxi 214122 Jiangsu, China
X. Ma (*) : A. Li
School of Biotechnology, and State Key Laboratory of Bioreactor
Engineering, East China University of Science and Technology,
Shanghai 214122, China
e-mail: maxy@ecust.edu.cn
H. Zhu
Center for Bioengineering and Biotechnology,
China University of Petroleum (East China),
Qingdao 266555, China

Introduction
The necessity of heterogeneously expressing more than one
gene spontaneously can be understood in the following two
aspects. First, structural and functional studies of many multiprotein complexes depend on the efficient over-expression of
these recombinant proteins in suitable hosts (Yokoyama 2003;
Cowieson et al. 2008; Bieniossek et al. 2009; Perrakis et al.
2011); second, investigations in metabolic engineering and
synthetic biologyan active research field that is attracting
increasing attention from biological scientistsfrequently
involves the expression of metabolic enzymes in the pathway
of interest (Kaznessis 2007; Purnick and Weiss 2009; Na et al.
2010; Nandagopal and Elowitz 2011; Vick et al. 2011). With
the help of restriction endonuclease (RE) EcoR I and ligase
from E. coli, Cohen and colleagues performed the first plasmid construction in 1973 (Cohen et al. 1973). Since then,
digestionligation-based genetic operations have been one of
the most straightforward and effective practices in recombinant plasmid construction. One can easily insert a gene of
interest into any available plasmid vector if a restriction
enzyme can cut the vector and cannot cut the heterogeneous
gene. The usual incompatibility of the ends generated by
different restriction enzymes makes it possible to insert a
foreign fragment with the desired orientation in the plasmid
by digesting the gene of interest and vector with two different
restriction enzymes. However, the increase in the length of the
inserted sequences will lead to a significant decrease in the
number of usable restriction enzyme sites. Therefore, a plasmid vector suitable for the expression of one gene will be less
effective (if not entirely useless) due to the availability of
restriction enzyme sites in studies of structural biology, metabolic engineering, and synthetic biology, in which the spontaneous expression of multiple genes in one host is frequently
involved.

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Therefore, in metabolic engineering and synthetic biology study, methods that can efficiently assemble multiple
expression cassettes for the construction of the desired pathway in model microorganisms are highly desired (Wingler
and Cornish 2011). This conclusion can be justified by the
following facts: First, the traditional classic digestion and
ligation method depending on the available restriction
enzyme sites cannot fulfill the task of constructing recombinant plasmids with multiple inserts (Busso et al. 2011).
Second, at least currently, the investigations in metabolic
engineering and synthetic biology can mainly follow a trial
and error process (Carrera et al. 2009; McArthur and Fong
2010; Lee et al. 2011). It is difficult to succeed in constructing a pathway, which is tested to be efficient by following
only one theoretically designed reasonable pathway
(Purnick and Weiss 2009). From this sense, the current
metabolic engineering and synthetic biology are much similar to the case of protein engineering: Rational design of
mutant protein is more promising, and it is the direction that
protein engineering will go; however, many enzymes with
improved properties are frequently obtained by directed
evolution in which an essentially identical trial and
error technical route is followed. Therefore, several
trials (optimization) might be necessary. Although a
reasonable design will make it possible to get the
expected construct with fewer trials and errors, materializing the design by rationally putting suitable genetic elements together is always an indispensible step and cannot be
circumvented in investigations in metabolic engineering and
synthetic biology nowadays (over-expression of metabolic
enzymes is frequently involved) and in the future (suitable
expression for robust circuit operation and balanced metabolic
redistribution are more popular). If there is no effective method for constructing the different designs of the pathways, it
will be impossible to obtain a satisfactory one (Wingler and
Cornish 2011).
In this review, we will summarize the available methods,
which can be used for assembling multiple expression cassettes for metabolic engineering and synthetic biology
research, with the intention of providing a comprehensive
reference for readers. These methods include the employment of a compatible multiple plasmid system, rarecutter-based digestion and ligation, in vitro recombination embodied by sequence and ligation independent
cloning, and in vivo recombination utilizing yeast or
the Cre-loxP system.

Employment of a compatible multiple plasmid system

Cloning a gene into a suitable plasmid through digestion and
ligation is the classic method for over-expressing it. Unfortunately, it is quite inconvenient to express more than one

Appl Microbiol Biotechnol (2012) 93:18531863

gene in most plasmids by constructing the recombinant

plasmid in this way. To simplify the over-expression of a
few genes in one cell, a plasmid with more than one promoter will be the first choice (Kim et al. 2004). Novagen
provides a set of plasmids featured by two multiple cloning
sites. These plasmids also carry different antibiotic-resistant
genes, conferring the recombinants corresponding resistance to the given antibiotics. The replication origins of
these plasmids are designed to be compatible for the peaceful co-existence of these plasmids in a cell (visit http://www.
merck-chemicals.com for details) which is generally
regarded as the premise for stable co-expression, although
theoretically incompatible plasmids have also been successfully used for co-over-expression (Yang et al. 2001). With
the most common expression host E. coli BL21(DE3), the
four plasmids can realize the expression of eight foreign
genes (2 genes 4 plasmids) spontaneously. This provided a very simple solution for the co-expression of
several proteins (Wladyka et al. 2005). However, the
maintenance of a stable co-existence requires the presence of a maximum of four antibiotics in the culture
medium. Although the concentration of each antibiotic
is only half of that in routine use, the antibiotics will also
constitute a physiological burden for the growth of recombinant cells (Dennis et al. 1985).

Employment of rare cutters for recombinant plasmid

construction
Despite the fact that there are several restriction sites in the
multiple cloning site of any available plasmid, the number
of usable sites will decrease sharply after the insertion of a
gene into the multiple cloning site, except for certain vectors
(Scheich et al. 2007). The newly introduced sequence will
inevitably contain some restriction sites, and restriction sites
in the multiple cloning sites are commonly found. Wakamori et al. developed a series of vectors. These plasmids
contain a set of rare-cutter sites, into which independent
expression cassettes can be subcloned. In the development
of these plasmids, the authors firstly decreased the size of
the pET15b to a smaller plasmid while the expression efficiency remained unaffected, by deleting certain parts of the
plasmid. At the same time, some restriction sites were also
removed by site-directed mutagenesis. Secondly, a sequence
containing nine rare cutters (Swa I, Asc I, Sbf I, Fse I, Sfi I,
Rsr II, Not I, Pac I, and Pme I) was inserted, forming a
multiple cloning site for the co-expression of multiple
genes. With this vector system, a seven-subunit protein
complex composed of the mammalian 26S proteasome regulatory subunits RPT1 to RPT6 and their associated factor
gankyrin was successfully co-expressed and co-purified as
confirmed by western blotting (Wakamori et al. 2010).

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Ligation independent cloning (LIC) (Aslanidis and de Jong

1990) can be regarded as the prototype of the in vitro recombination method to be discussed here because LIC is dependent on some specially designed plasmid vectors. Some
companies (e.g., Novagen) commercially provide some plasmid vectors that are designed for LIC. In this kind of plasmid,
the recognition sequences for a certain restriction enzyme that
will generate blunt ends are included in the multiple cloning
site section. On both sides, the restriction site is flanked by a
short sequence, which is designed in such a way that one type
of nucleotide is absent. The target insert is amplified by
polymerase chain reaction (PCR) with primers containing
the homology sequence to the flanking sequences in the
plasmid. When cloning, the plasmid is linearized with a given
enzyme and subjected to the treatment of T4 polymerase in the
presence of dNTP that is absent from the flanking sequences.
Taking advantage of the biochemical properties, mainly the 3
to 5 exonuclease activity of T4 DNA polymerase, the two
ends of the PCR product and the linearized plasmid are
degraded from the 3 ends to a base that is identical to the
dNTP added to the system, resulting in a 5 overhang. Since
there is homology between the PCR product and the linearized
plasmid, the single-stranded ends can anneal each other to
form double strands that are stabilized by complementarity. A
recombinant plasmid is constructed. Actually, it is not a

covalently closed circular DNA (cccDNA) molecule. It carries

four nicks because of the design of the LIC plasmid. After the
plasmids have been transformed into chemo- or electrocompetent E. coli cells, these nicks will be repaired by linking
the two adjacent nucleotides with E. coli ligase. The same
strategy has also been employed in commercialized kit QuickChange from Invitrogen and another cloning method megaprimer PCR of whole plasmid (MEGAWHOP) invented by
Miyazaki and Takenouchi (Miyazaki and Takenouchi 2002;
Miyazaki 2011). The principle and procedure of LIC are
shown in Fig. 1.
Before transforming into competent cells, the recombinant plasmid is only stabilized by base pairing forces in the
two homologous parts of the PCR products and the linearized plasmid vectors. It is understandable that the recombinant plasmid is weaker than the recombinant plasmid
generated with ligation. Some conditions (e.g., the heat
shock involved in the transformation of plasmid into
chemo-competent cells) may break the molecule down into
two separate parts. According to Aslanidis report, a minimal homology length of 10 base is sufficient for satisfactory
LIC results, and a heat shock at a lower temperature (37 C)
for a longer period (5 min) is also workable for LIC (Aslanidis et al. 1994). However, it will be better to use LIC with
a longer homology tail and transform the recombinant plasmid prepared with LIC with electroporation, in which no
heat shock of a higher degree is involved. At the same time,
a higher efficiency of transformation can be easily obtained
through electroporation.

Fig. 1 Principle and procedure of ligation independent cloning (LIC).

A gene of interest is amplified with additional sequences attached to its
5 end, producing the PCR product ready for LIC (I). Treatment of this
PCR product with T4 DNA polymerase in the presence of dATP
generates single-strand overhangs (II). The presence of dATP ensures
that the degradation of double-stranded DNA by T4 DNA polymerase
stops at the site occupied by an adenine base (A). A linearized LIC
vector (here, the sequence is from pET-32 Xa/LIC plasmid vector) is
subjected to T4 DNA polymerase treatment under the same conditions

(III). Mixing the two DNA samples (with different molar ratios) in
buffer favoring the annealing between complementary single-stranded
DNA promotes the formation of recombinant plasmids with nicks.
These are repaired by the DNA repairing system in E. coli cells,
resulting in covalently closed circular DNA plasmids. In the figure,
the four small solid arrows indicate where nicks have been formed in
the DNA molecule. The two parts inside the box (dotted line) depict
regions that stabilize the recombinant plasmid before being transformed into competent cells

In vitro recombination
Ligation independent cloning

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As mentioned previously, LIC is dependent on a specially

designed plasmid vector. Li and Elledge (2007) invented
another method which is essentially similar to LIC, but
which enables the cloning of foreign fragments into any
desired region of a vector; this is termed sequence and
ligation independent cloning (SLIC). It harnesses the in
vitro recombination of the single-stranded overhangs generated by T4 DNA polymerase treatment of the PCR product

and the vector. Under this method, the circular plasmid

vector should also be linearized. In general, there are two
ways to prepare the linearized vector: restriction enzyme
digestion and PCR amplification. For a given plasmid vector,
any enzyme listed in the MCS can be used to linearize the
vector for SLIC if the regulatory components (promoter, ribosome binding site, transcription terminator, etc.) designed in
the plasmid are to be used for the expression of the foreign
gene. Digestion with two enzymes producing incompatible
ends will minimize the false-positive transformants in the

Fig. 2 Principle and procedure of sequence and ligation independent

cloning (SLIC) and spontaneous cloning of multiple fragments with
SLIC. a Principle and procedure of sequence and ligation independent
cloning (SLIC). A target gene is amplified using PCR with specially
designed primers which contain the sequences homologous to the
vector being used (depicted in bold, 1). This generates a PCR product
with additional sequences for SLIC (2). After being treated with T4
DNA polymerase, single-stranded terminals are produced (3). The
plasmid that is to be used for cloning is digested with single (or double)
enzymes (4) or amplified using primers designed to generate PCR
product with ends that share homology to the ends of PCR product
of the target gene (5). T4 DNA polymerase treatment is used to
generate single-stranded overhangs, used for annealing with the PCR

product of the target gene (6). Mixing the DNA samples (3) and (6) in
ligation buffer enables the formation of recombinant plasmids with
nicks (7), which are subsequently repaired by E. coli enzymes after
transfer into competent cells (Li and Elledge 2007). b Spontaneous
construction of recombinant plasmids containing multiple fragments
with SLIC. Multiple fragments to be inserted into the same plasmid
vectors are amplified as products with ends homologous to each other,
using specially designed primers. Treatment with T4 DNA polymerase
generates single-stranded overhangs compatible with intermolecular
annealing. Mixing these products with linearized plasmid vectors in a
suitable buffer encourages the formation of recombinant plasmid in
which the homologous ends combine to ensure that all the fragments
assemble in the correct order

Sequence and ligation independent cloning

Appl Microbiol Biotechnol (2012) 93:18531863

selective agar plate due to the self-circularization of the plasmid vector, although linearization with one enzyme is also
workable (Fig. 2). In SLIC, if the plasmid vector is linearized
with restriction enzyme digestion, incomplete digestion and
the self-circularization of the linearized plasmid might be the
main factors that lead to false-positive resistant colonies on
selective plates.
If PCR amplification is used to generate a linearized
plasmid vector, the target gene can be inserted into the
vector at any desirable site. However, the expression of
foreign genes inserted in the region beyond the ranges
defined by the designed MCS will require the necessary
regulatory elements, such as promoter, transcription terminator, etc. Because the plasmid is used as the template in
PCR, a subsequent treatment with Dpn I that will specifically degrade the methylated DNA should be carried out to
eliminate the false-positive transformants resulting from the
template plasmid.
With the SLIC method, the cloning of foreign sequences
into any sites of a vector can be realized. Thus, this can be
used to express more genes in one plasmid, which is routinely
used for the expression of one gene. This is very important,
especially for synthetic biology study. As reported by Li and
Elledge (2007), the PCR product amplified with a modified
PCR thermocycling program in favor of the generation of an
incomplete PCR product by omitting the last extension step
for several minutes also provided the ready materials for
SLIC. Meanwhile, an acceptable target gene fragment with
single-stranded ends could also be obtained by mixing and
reannealing the PCR products of the target gene amplified
with two set of primers, which would generate a product with
a 5- or 3 additional sequence, respectively (Tillett and Neilan
1999) (Fig. 3).
SLIC can be used to insert multiple fragments of DNA
into a desirable plasmid vector in an expected order when
these fragments were prepared in such a way as to make
them capable of carrying sequence homology to each other.
One can use necessities from different suppliers to carry out
the SLIC. In addition, several commercial kits have been
developed by different companies, for example, In-fusion
from Clontech, CloneEZTM from GenScript, GeneArt seamless cloning and assembly from Invitrogen, etc. The necessities of SLIC were provided as a mixture in the form of dry
powder or solution in an optimized setting for high efficiency.
The recA enzyme can promote the annealing between DNA
samples of low concentration ready for SLIC; improved efficiency can also be obtained by increasing the concentration of
each DNA fragment.
Notably, simply by placing the encoding sequences one
after another, it is very easy seamlessly to clone them together
in a defined order with these commercialized SLIC kits
because the 5-terminal of one gene is usually different from
the 3-terminal of another gene. The homology part of these

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Fig. 3 Generation of PCR product with single-strand overhangs for

SLIC. A target gene is amplified with two sets of primers (P1F-P1R
and P2F-P2R). In each pair of primers, only one of the primers carries
an additional sequence in the forward or reverse direction (A). The
primer pair P1F-P1R generates a PCR product with an additional
sequence at the 5 end (B), while P2F-P2R has an additional sequence
at the 3 end (C). Denaturation by heating and annealing with gradual
cooling after mixing the two PCR products together facilitates the
formation of molecules with 5 overhangs (theoretical yield 25%) that
are ready for SLIC (Tillett and Neilan 1999; Li and Elledge 2007)

PCR products can guarantee the right order in assembly.

However, the efficient expression of these encoding sequences
assembled is the main purpose of these investigations. Therefore, other elements (promoter, ribosome binding site, terminator, etc), in some cases, should be added at the 5- or 3terminal of the encoding sequences. The conserved parts of
these elements might make it far more complicated in primer
design to realize the expected order in assembly as defined by
homology in the ends, although the adoption of a linker might
be helpful (Ramon and Smith 2011).

One-step isothermal in vitro recombination

This method is very similar to ligation independent cloning,
which depends on the annealing of single-stranded ends
generated by exonuclease treatment. The difference is that,
in ligation independent cloning, the ends of the DNA molecules carry 5 overhangs because the 3-exonuclease activity of
T4-polymerase is employed. In isothermal in vitro recombination, the 5-exonuclease activity of heat liable T5 is used to
produce the overhangs. In this method, the homology sequences required for subsequent annealing are placed at one end of
each substrate DNA molecule prepared by PCR amplification
or excision from recombinant vectors. The degradation by the
T5 exonuclease of each strand from the 5 end produces
3 overhangs at each terminal. The reaction is at an optimized
temperature of 50 C. Because the T5 exonuclease is heat
liable, it will be inactivated during incubation at this temperature. The heat-resistant Phusion DNA polymerase and Taq
ligase remain active, and they will fill the gaps in the newly
formed molecules due to annealing in the single-stranded

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Fig. 4 One-step isothermal in

vitro recombination. At 50 C,
DNA samples prepared for
isothermal in vitro
recombination (A) were
mixed with heat liable T5
exonuclease, thermostable
Phusion polymerase, and
Taq ligase. The T5 exonuclease
degrades the DNA molecules
from 5-terminal, producing
single-stranded ends ready for
in vitro recombination (B). T5
exonuclease is heat sensitive and
becomes inactivated after
a period of incubation at 50 C
(C). Thermostable Phusion DNA
polymerase and Taq ligase work
together to repair the gaps after
single-strand annealing (D)
(Gibson et al. 2009)

homology regions, making it a covalently linked recombinant

molecule (Gibson et al. 2009) (Fig. 4).

In vivo recombination
Yeast-based in vivo recombination
Unlike in vitro recombination, which requires the artificially
generated single-stranded ends of the substrate DNA molecules, in vivo recombination needs only the homology parts
with a reasonable length among the DNA fragments to be
joined together, greatly simplifying the process. The expression of multiple proteins can be achieved by constructing
the desired DNA sequences through in vivo recombination
from smaller fragments. For its relative simplicity and the availability of various methods for genetic operations,
recombination-efficient microorganism yeast is the first choice
for in vivo recombination(Krivoruchko et al. 2011). As one of
the model organisms, it is regarded as the E. coli of eukaryotes in
researches. Many shuttle vectors can rapidly propagate in E. coli
and efficiently integrate into the genome. In 1987, Ma et al.
reported the method for plasmid construction by homologous
recombination in yeast (Ma et al. 1987), a useful method that
was later generalized by Raymond et al. (Raymond et al. 1999).
In 2001, Gunyuzlu et al. proposed the linker-assisted homologous recombination for plasmid construction in yeast (Gunyuzlu
et al. 2001). The super recombination capability of yeast makes
it possible to assemble multiple fragments of it into one plasmid
spontaneously, greatly simplifying the steps when one gene has
to be expressed in different hosts, and other features of the

expressed protein are also expected (e.g., some tags) (Raymond

et al. 1999). It can also be used to construct one long DNA
fragment from shorter ones. If mutations are incorporated into
these short fragments, multiple mutations of the target gene can
be achieved in one recombination step (Fig. 5).
Actually, yeast has been used for the one-step assembly
of the complete genome of Mycoplasma genitalium
(~592 kb) from 6 (Gibson et al. 2008a) or 25 (Gibson et
al. 2008b) overlapping DNA fragments, and the even larger
genome (~1,114 kb) of Mycoplasma mycoides, which has
been proven to be functional in controlling a cell (Gibson et

Fig. 5 Assembling shorter fragments carrying mutations that can undergo

in vivo recombination into a full-length gene. It is possible to assemble a
gene from a number of fragments; each of which carries a small number of
desired mutations. The ends of each fragment are designed with overlaps
that enabled the correct order of assembly in recombination. Cotransformation of these fragments with linearized plasmids and subsequent
in vivo recombination will lead to the formation of recombinant plasmids
harboring the desired mutant gene containing multiple mutations

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al. 2010). These good examples have sufficiently demonstrated the feasibility of using an in vivo recombination
of yeast to DNA fragment that can express multiple
proteins for synthetic biology. As a powerful assembling
machine, the limitations of the assembly methods in
yeast remain unknown (Gibson et al. 2008b). However,
the recombination taking place in vivo requires the cell
to take up enough DNA fragments, and the required
amount of DNA for successful assembly will increase
when a larger final assembled product is expected. This

might be the reason why the proplast of yeast was used

in these experiments on genome assembly.
In addition to assembling foreign DNA fragments
with overlaps into independent larger DNA molecules,
the in vivo recombination of yeast has also been explored for the successive integration of foreign DNA
fragments into a large expression unit in the yeast
genome (Wingler and Cornish 2011). Wingler et al.
termed this method reiterative recombination, based
on the fact that double-strand breaks of DNA can

Fig. 6 General scheme of reiterative recombination. A yeast acceptor

host is created by integrating a DNA construct containing a promoter
(prom), a selective marker (depicted as marker 2), a recognition sequence
homing endonuclease (endonuclease 1), and a homology region for
recombination (A). The first round of reiterative recombination is initiated
through the introduction of another plasmid (shown as a circle) carrying
the gene of interest (gene 1) through transformation. The key elements in
the plasmids are endonuclease 1 (whose expression is inducible), marker
1, a recognition sequence for another homing endonuclease (endonuclease 2), and the downstream sequence for recombination (B). After adding
an inducer, the homing endonuclease 1 (endonuclease 1) was expressed.
The expressed endonuclease 1 recognizes and cleaves a sequence in the
yeast chromosome, producing a double-strand break, open for recombination to occur in the homologous regions (C). Selection based on the

expression of maker 1 identified the positive recombinants containing the

newly integrated gene (gene 1). At the same time, the recognition
sequence for homing endonuclease 2 was also introduced (D). Integrating
a second gene (gene 2) is initiated by the transformation of the second
plasmid, in which there is endonuclease 2 (endonuclease 2), marker 2,
and a recognition sequence for endonuclease 1 (E). Induced expression of
endonuclease 2 leads to the breakage of the chromosome at the site for
endonuclease 2, introduced through the integration of the first plasmid
(F). Another selection based on marker 2 results in the identification of
recombinants harboring the gene (gene 2) introduced during the second
integration (G), which is also the starting acceptor host for a new round of
integration through homologous recombination (this figure was redrawn
according to Fig. 1 in Wingler and Cornish 2011

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promote homologous recombination for DNA repair in

yeast (Storici et al. 2003). In reiterative recombination,
the efficiency of the recombination was significantly
improved by induced breakage due to the expressed
homing endonuclease. Two recyclable markers functioned in an alternate way, enabling the identification
of positive recombinants through selection for successively assembling DNA constructs at the expected site
of the chromosome. The number of expression units
integrated into the chromosome was theoretically infinite
because the process was iterative and repeatable
(Fig. 6). The new method had been demonstrated to
be feasible for the construction of a biosynthetic pathway library with a size of 104. This was of especial
significance give the fact that a rational design of a
satisfactory multiple-gene pathway was still relatively

Fig. 7 Multiplication-module-mediated assembly of multiple gene

expression cassettes ready for Cre-loxP site-specific recombination
into Bacmid. Two plasmids, pUCDM and pFBDM, are constructed
to harbor the same multiplication module (M), in which the sites of
BstZ 17I, Spe I, Cla I, and Nru I are compatible with Pme I and Avr II
flanking the two MCS sites (MCS1, MCS2). Cloning two genes into
one of the two plasmids (I or III) can be achieved through digestion and
ligation by choosing a suitable restriction site in the multiple cloning
sites, which generates a recombinant plasmid with two independently
expressible cassettes (AB in plasmid II or CD in IV). Releasing the

Appl Microbiol Biotechnol (2012) 93:18531863

difficult for and inaccessible to most researchers, casting

a wider net and fishing out the best one from a pathway
library, simply as the principle of directed evolution
(T. W. Wang et al. 2006) and adaptive laboratory evolution
(ALE) (Portnoy et al. 2011).
The Cre-loxP site-specific recombination-based system
Researchers have developed a number of different methods
that take advantage of the Cre-loxP site-specific recombination system to fulfill the demand of multiple protein expression. This system has even been used to construct an entire
yeast chromosome (Dymond et al. 2011). Berger et al. (2004)
constructed two derived plasmids (pUCDM and pFBDM)
with pUNI10 and pFastBacDUAL as the individual template
(Fig. 7). Two multiple cloning sites were placed into each

expression cassettes (AB or CD) through digestion by Pme I and Avr

II and then ligating it with a second recombinant plasmid (IV) digested
with suitable enzymes in the multiplication module complete the
transfer of a two-gene expression cassette (AB) into the second
recombinant plasmid, which expresses a further two genes (CD).
The new recombinant plasmid (V) can express four genes independently. Continuing the process enables the insertion of multiple genes, with
two new genes incorporated each cycle (this figure was based on Fig. 1
in Berger et al. 2004)

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plasmid (MCS1 and MCS2), facilitating digestionligationbased cloning. Expression of foreign genes inserted into these
sites was controlled by the promoter p10 or polh. The two
expression cassettes were flanked by two unique restriction
sites, Pme I and Avr II, used to release the combined expression cassettes together from the recombinant plasmid. The
restriction enzymes have compatible ends (Pme I, BstZ 17I,
and Nur I; Avr II and Spe I), enabling the released double
cassette to be further inserted into another recombinant plasmid that already contains another two genes, digested by
enzymes whose sites are located in the multiplication module
(a sequence with BstZ 17I, Spe I, Cla I, Nru I sites). After this,
a recombinant plasmid containing four genes in independent
expression cassettes (each gene has its own promoter and
terminator) has been constructed. In this new recombinant
plasmid, the sites for Pme I and Avr II are retained and can
be used for transferring this four-gene fragment into a further
recombinant plasmid in the same way. The authors demonstrated that multiple gene expression cassettes constructed
using this method could be inserted into the MultiBac bacmid
by Cre-loxP site-specific recombination (Fitzgerald et al.
2006). In combination with the use of Tn7 transposition, it is
possible to achieve expression of mutant protein complexes
with different subunits, or modification of an expressed protein can be achieved.
In 2009, Bieniossek et al. described the Acembl system:
automated unrestricted multigene recombineering to achieve

the production of multiprotein complexes (Bieniossek et al.

2009) (see Fig. 8). The procedure that they describe can be
regarded as an improved version of the Cre-loxP-mediated
site-specific recombination procedure, described previously.
The Acembl system is primarily composed of three donor
vectors and two acceptor vectors. The difference between
these vectors is the replication origin, the antibiotic resistance, and the promoter (and corresponding terminator) sequences. All vectors share a common identical
sequence of multiple integration elements (MIE) and a
common loxP portion for Cre-loxP-mediated site-specific
recombination. Foreign genes can be inserted into the
MIE through digestion and ligation or sequence and
ligation independent cloning methods. Transferring or
inserting one expression cassette (containing one or more
genes) from one vector to another can be achieved by ligating
the expression cassettes released (using homing endonuclease
and Bst XI) with a vector linearized with a homing endonuclease. This is possible due to the compatibility between the ends
produced. As well as through ligation, constructing a recombinant plasmid containing multiple expression cassettes can
also be carried out with Cre-loxP site-specific recombination.
When incubating and fusing recombinant vectors with the Cre
enzyme, all possible combination between these vectors will
occur. Desired positive recombinants can subsequently be
screened through the use of antibiotic resistance or propagation
in suitable host.

Fig. 8 The plasmid used for multiple protein expression in Acembl. A

set of five small plasmids (acceptor vector: pACE and pACE2; donor
vector: pDC, pDK, and pDS) is specially designed through the elimination of unnecessary sequences. These plasmids contain the identical loxP
sites for Cre-loxP-mediated site-specific recombination and multiple
integration elements (MIE). Different replication origins, antibiotics
resistance, and promoters (pACE: oriBR322, ampicillin (Ap), T7; pACE2:
oriBR322, tetracycline (Tet), T7; pDC: oriR6K, chloramphenicol (Cm),
T7; pDK: oriR6K, kanamycin (Kn), lac; pDS: oriR6K, spectinomycin
(Sp), lac) are incorporated into these plasmids for subsequent selection
and expression of the gene of interest. One or several genes can be
inserted into the MIE site through digestion and ligation or sequence
and ligation independent cloning methods. The homing endonuclease site

(HES, acceptor vectors: I-CeuI and donor vector: PI-SceI) and Bs tXI are
used as flanking regions from promoter to terminator. The ends generated
by the I-Ceu I, PI-Sce I, or Bst XI are compatible, enabling the insertion of
multiple expression cassettes released with the homing endonuclease
enzyme and Bst XI, into the vector linearized by the homing endonuclease enzyme (I-Ceu I, PI-Sce I). Joining of the donor and acceptor vectors
can be achieved by Cre-loxP meditated site-specific recombination. Mixing recombinant donor and acceptor vectors in the presence of the Cre
enzyme promotes the formation of a fused plasmid. Positive fusion
plasmids can be selected based on replication origin (the donor vector
can only be propagated in pir expressing host) and antibiotics resistance
(Bieniossek et al. 2009)

1862

Spontaneous modification of multiple targets on genome

with in vivo recombination
The aforementioned methods are related to the construction of genetic systems by introducing desirable genes
into a plasmid or into a genome. Wang et al. invented a
method called multiplex automated genome engineering
(MAGE) (H. H. Wang et al. 2009). This method enabled the spontaneous and continual modification of
multiple targets on genomes of a population of living
cell. Briefly, the growing cells (usually at the exponential stage) to be engineered are prepared into electrocompetent cells. Synthetic oligos were added into the
suspension of competent cells and introduced into cells
by electroporation. Inside the living cells after electroporation, oligos would promote the recombination in the
region specified by the sequences at the ends of the
introduced oligos through allelic replacement. This
method offers a way to modify genome with a high
throughput. Moreover, it successfully circumvents the
assembling of genes into a synthetic pathway. An indispensable premise for the successful application of this
method is the existence of target pathway in the organism in operation.

Conclusions and prospects

Digestion and ligation based genetic operations have
contributed tremendously to modern biotechnology.
However, these techniques are insufficient for structural
elucidation of multicomponent protein complexes and
investigations in synthetic biology. Recently, development of novel recombination techniques (both in vitro
and in vivo) and modification/enhancement of conventional cloning techniques have greatly enriched the
available toolbox. The ultimate overall behavior of a
constructed cassette is directly determined/affected by
subsequent processes (such as transcription, translation
and posttranslational modification, folding and assembly
into functional protein molecules) that take place in the
expression process under known/unknown regulations in
the host. For this reason, it is of great importance, as an
alternative solution, to provide different designs of expression cassettesthe templates for transcriptionprepared using methods suitable for high-through expression
cassette construction. This is especially true when the rational
design of multiple expression cassettes is not quite accessible
for many researchers.
Acknowledgements Grants from the National Science Foundation of
China (31000054, 30873190) and the Fundamental Research Funds for
the Central Universities (No. JUSRP10917) supported this research.

Appl Microbiol Biotechnol (2012) 93:18531863

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