| In site-specific recombination, a phage encoded recombinase interacts with the attachment site of bacterial (attB) and of phage (attP), brings the sites together in a synapse, and then catalyzes strand exchange so that this DNA is cleaved and relegated to the opposite partners; leaves the phage in lysogenic cycle. The integration and excision recombinations between bacterial phage?and its host E.coli were considered as the research model for the molecular mechanism of site-specific recombination. Site-specific recombination describes a variety of specialized recombination processes from different organisms with particular functions. Generally, site-specific recombination includes:phage integration and excision with the host chromosome(E.coli phage?and Streptomyces phage?C31); the resolution of cointegrates derived from transposition (transposon??and Tn3); some gene activation and regulation events (the DNA inversions responsible for flagellar phase variation in Salmonella typhimurium). Just as its name implies, site specific recombination occurs between specific sites; the tyrosine or serine in the active center of recombinases attacks the DNA sugar-phosphate backbone to mediate double strand cleavage and exchange.Site-specific recombinases have been widely used in genetic engineering; the advantages, of high substrates specificity and efficiency, rapid kinctics and convenience to modify, have greatly accelerated the transformation of SSRs into daily laboratory use. As a collection of highly efficient enzymes, a series of site-specific recombination systems and their derivates were used in various model organisms; Cre-loxP of phage P1, Flp-frt of yeast plasmid 2?,?C31-att of Streptomyces phage were most frequently used systems. Innovative and creative modifications of these systems are ongoing to fully exploit their advantages; and new SSR systems were constantly developed, such as Bxbl and Dre recombinases. Urbanski and Condie from University of Georgia developed an online server for data mining of literatures about SSR systems (http://ssrc.genetics.uga.edu/); researchers could easily find nearly ten thousand papers of structural and applied studies in bacterial, Drosophila, zebra fish, stem cell, Arabidopsis thaliana etc., through this online server.In this study, we established a highly efficient site-specific recombination system based on Streptomyces phage?BT1, and exploited the molecular mechanisms of the integrase catalyzed reactions as well as opened up the possibility of application in synthetic biology. The work was carried out as follows:Firstly, the enzymatic activity of the?BT1 integrase was characterized in vitro. We showed that this integrase has efficient integration activity with substrate DNAs containing attB and attP sites, independent of DNA supercoiling or cofactors. Both intra- and inter-molecular recombinations proceed with rapid kinetics. The recombination is highly specific and no reactions are observed between pairs of sites including attB/attL, attB/attR, attP/attL, attP/attR, or between two identical att sequences; however, a low but significant frequency of excision recombination between attL and attR is observed in the presence of the?BT1 integrase alone. In addition, for efficient integration, the minimal sizes of attB and attP are as 36 bp and 48 bp, respectively.Integration of?BT1 and?C31 via their attachment (att) sites is catalyzed by integrases of the large serine recombinase subtype. Both?BT1 and?C31 integrases were found to cleave single substrate att sites without synaptic complex formation, and?BT1 integrase relaxed supercoiled DNA containing a single integration site. Systematic mutation of the central att site dinucleotide revealed that cleavage was independent of nucleotide sequence, but rejoining was crucially dependent upon complementarity of the cleavage products. Recombination between att sites containing dinucleotides with antiparallel complementarity led to antiparallel recombination. Integrase-substrate preincubation experiments revealed that the enzyme can form an attP-integrase tetramer complex that then captures naked attB DNA, suggested two alternative assembly pathways can lead to synaptic complex formation.Since the core di-nucleotides of recombination sites were not participated in DNA recognition and binding; we mutated the core-sequences and obtained 16 pairs of non-compatible recombination sites. Combined with the high efficiency of?BT1 integrase-mediated site-specific recombination reactions, we developed a highly efficient and accurate method for multiple module assembly in one simple reaction, which designated as "Site-Specific Recombination based Tandem Assembly (SSRTA)" method. The biosynthetic gene cluster of epothilones, which is the most promising anti-tumor agents, was chosen to illustrate this method. We successfully constructed 5 and 7 DNA segments into circular DNA in one-step and tandem manner, the largest assembled products was 62.4 kb DNA molecular in our tests. This SSRTA method described here provided an efficient and accurate route to the combination and assembly of related genetic pathways, the deletion and substitution of combined modules for synthetic biology.In conclusion, we firstly established a highly efficient site-specific recombination system based on Streptomyces phage?BT1, and extensively studied the molecular mechanisms of recombination reactions through biochemical strategies; these data revealed new insight into the mechanisms of DNA cleavage and synapsis formation during site-specific recombination mediated by bacteriophage-encoded large serine recombinases. Further more, after selection of non-compatible recombination sites-pairs, we developed an efficient and accurate approach for multiple DNA segments assembly named SSRTA; further construction and modification of this method will bring a robust strategy for synthetic biology. |
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