Appl Microbiol Biotechnol (2012) 93:1853–1863 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:
[email protected] 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 biology—an active research field that is attracting increasing attention from biological scientists—frequently 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, digestion–ligation-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
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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
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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 3′terminal 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 (A–B in plasmid II or C–D in IV). Releasing the
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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 (A–B or C–D) 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 (A–B) into the second recombinant plasmid, which expresses a further two genes (C–D). 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 digestion–ligationbased 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: oriR6Kγ, chloramphenicol (Cm), T7; pDK: oriR6Kγ, kanamycin (Kn), lac; pDS: oriR6Kγ, 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)
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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 cassettes—the templates for transcription—prepared 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.
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