**4. Gateway technology for DNA cloning**

Gateway technology is another site-specific recombinational cloning method [54, 56] (**Figure 4**).- This technology is based on the site-specific recombination system of *E. coli* bacteriophage λ [57]. Βacteriophage λ integrates into the *E. coli* chromosome by site-specific recombination- between the attachment (*att*) sites on the *b*acterial chromosome (attB) and the *att* sites on the *p*hage chromosome (attP), to generate left (attL) and right (attR) att sites. This recombination-

**Figure 4.** Standard Gateway method for single-fragment cloning. The DNA fragment of a gene of interest is amplified by PCR with a cloned DNA or cDNA library as the PCR template. The destination clone containing a polyadenylation (polyA) tail region downstream from the cDNA can be used as a PCR template for the next MultiSite Gateway cloning. Abbreviations: Kan, kanamycin resistance gene; Amp, ampicillin resistance gene; Cm, chloramphenicol resistance gene.-

reaction is mediated by the integrase (Int) enzyme of bacteriophage λ and the integration host factor (IHF) of *E. coli.* The excision reaction requires another host factor, excisionase (Xis), in addition to Int and IHF.-The Gateway technology has developed mutant attsites (such as attB1,- attB2, attP1, and attP2) [56]. The attB1 site specifically recombines with the attP1 site, but not- attP2, to generate the attL1 and attR1 sites. Similarly, the attB2 site specifically recombines with- the attP2 site to generate the attL2 and attR2 sites.-

 In the first step of the Gateway cloning, the DNA fragment for cloning is amplified by PCR with- primer sets containing attB1 or attB2 sequences at the 5′ ends (**Figure 4**). Gateway cloning vectors (pDONR) contain attP1 and attP2 sites for cloning. Therefore, the PCR product containing- the attB1 or attB2 sites at both ends can be specifically inserted in between the attP1 and attP2- sites of the pDONR vector by site-specific recombination, to generate the entry clone of the- Gateway system. A protein mixture (BP clonase) containing Int and IHF is used for this *in vitro*  site-specific recombination reaction (BP reaction). In the pDONR vector, the *ccdB* gene from F- factor of *E. coli* is located between the attP1 and attP2 sites. The CcdB protein is a toxin for *E. coli* cells that lack the antitoxin, the CcdA protein, which is also produced from F factor of *E. coli*  [58–61]. Therefore, *E. coli* cells without F factor or the *ccdA* gene cannot grow, due to the production of the CcdB protein from the pDONR vector. For example, *E. coli* DH5α cells lack F factor- and the *ccdA* gene. Therefore, the cell growth of the DH5α strain is inhibited in the presence of the pDONR vector. The Gateway cloning method applies this cell killing mechanism mediated- by the *ccdB* gene for efficient DNA cloning. If the PCR product is successfully cloned between- the attP1 and attP2 sites of the pDONR vector, then the *ccdB* gene is removed from the vector- by the site-specific recombination. Therefore, when the *in vitro* site-specific reaction mixture- is transformed into DH5α cells, only the cells containing the generated entry clone can grow.- Thus, all of the transformed colonies contain the successfully cloned plasmid DNA.-This is an- excellent point of the Gateway cloning method. In the constructed entry clone, the cloned gene- is present between the generated attL1 and attL2 sites.-

 The Gateway cloning system provides destination vectors for numerous purposes, such as for- expressing the cloned gene in a variety of organisms. Each destination vector contains attR- sites for cloning on both sides of the *ccdB* gene (**Figure 4**). The cloned gene in the entry clone- can be transferred to destination vectors by an *in vitro* site-specific recombination reaction (LR- reaction) between the attL and attR sites with a protein mixture (LR clonase) containing Int,- IHF, and Xis, to generate the destination clone. As a result of the reaction, the *ccdB* gene of the destination vector is replaced with the gene of interest and is transferred to the pDONR vector- as a by-product. Therefore, when the LR reaction mixture is transformed into DH5α cells, the growth of the cells containing the by-product plasmid is inhibited by the expression of the toxic *ccdB* gene. The antibiotic resistance gene of the destination vector is different from that of- the pDONR vector. Therefore, only the desired destination clone is selected in the presence of- the appropriate antibiotics. Thus, once the DNA fragment of interest is cloned into the pDONR- vector, the DNA fragment can be transferred to a variety of destination vectors quite easily.-
