Synthetic genomics has provided new bottom-up platforms for the functional study of viral and microbial genomes.The construction of the large,gigabase(Gb)-sized genomes of higher organisms will deepen our understandin...Synthetic genomics has provided new bottom-up platforms for the functional study of viral and microbial genomes.The construction of the large,gigabase(Gb)-sized genomes of higher organisms will deepen our understanding of genetic blueprints significantly.But for the synthesis and assembly of such large-scale genomes,the development of new or expanded methods is required.In this study,we develop an efficient pipeline for the construction of large DNA fragments sized 100 kilobases(kb)or above from scratches and describe an efficient method for“scar-free”engineering of the assembled sequences.Our method,therefore,should provide a standard framework for producing long DNA molecules,which are critical materials for synthetic genomics and metabolic engineering.展开更多
Baculoviruses are large DNA viruses which have been widely used as expression vectors and biological insecticides.Homologous recombination and Bac-to-Bac system have been the main methods for manipulating the baculovi...Baculoviruses are large DNA viruses which have been widely used as expression vectors and biological insecticides.Homologous recombination and Bac-to-Bac system have been the main methods for manipulating the baculovirus genome.Recently, we generated a synthetic baculovirus Ac MNPV-WIV-Syn1 which fully resembled its parental virus Autographa californica multiple nucleopolyhedrovirus(Ac MNPV). Here, we report the modification of Ac MNPV-WIV-Syn1 into a novel bacmid, Ac Bac-Syn, which can be used as a backbone for Bac-to-Bac system. To achieve this, a vector contained a Lac Z:att Tn7 and egfp cassette was constructed, and recombined with a linearized Ac MNPV-WIV-Syn1 genome by transformation-associated recombination in yeast to generate bacmid Ac Bac-Syn. The bacmid was then transfected to insect cells and the rescued virus showed similar biological characteristics to the wild-type virus in terms of the kinetics of budded virus production, the morphology of occlusion bodies, and the oral infectivity in insect larvae. For demonstration, a red fluorescent protein gene Dsred was transposed into the att Tn7 site by conventional Bac-to-Bac method, and the transfection and infection assays showed that Ac Bac-Syn can be readily used for foreign gene insertion and expression.Ac Bac-Syn has several advantages over the conventional Ac MNPV bacmids, such as it contains an egfp reporter gene which facilitates visualization of virus propagation and titration;its DNA copy numbers could be induced to a higher level in E. coli;and the retaining of the native polyhedrin gene in the genome making it an attractive system for studying the functions of gene related to occlusion body assembly and oral infection.展开更多
基金supported by the National Key Research and Development Program of China(2018YFA0900100 and 2019YFA0903800)the Strategic Priority Research Program of the Chinese Academy of Sciences(XDPB18)+3 种基金the National Natural Science Foundation of China(31800069,32030004,31725002 and 32001065)Shenzhen Science and Technology Program(KQTD20180413181837372)Guangdong Provincial Key Laboratory of Synthetic Genomics(2019B030301006)Shenzhen Outstanding Talents Training Fund and the CAS President’s International Fellowship Initiative(2021VBB0002)。
文摘Synthetic genomics has provided new bottom-up platforms for the functional study of viral and microbial genomes.The construction of the large,gigabase(Gb)-sized genomes of higher organisms will deepen our understanding of genetic blueprints significantly.But for the synthesis and assembly of such large-scale genomes,the development of new or expanded methods is required.In this study,we develop an efficient pipeline for the construction of large DNA fragments sized 100 kilobases(kb)or above from scratches and describe an efficient method for“scar-free”engineering of the assembled sequences.Our method,therefore,should provide a standard framework for producing long DNA molecules,which are critical materials for synthetic genomics and metabolic engineering.
基金This work was supported by the grants from the National Science Foundation of China(Grant Nos.31800143 and 31872640)the Key Research Program of Frontier Sciences of the Chinese Academy of Sciences(Grant No.QYZDJ-SSW-SMC021)the Hubei Provincial Innovation Center of Agricultural Sciences and Technology(Grant No.2019-620-000-001-017)。
文摘Baculoviruses are large DNA viruses which have been widely used as expression vectors and biological insecticides.Homologous recombination and Bac-to-Bac system have been the main methods for manipulating the baculovirus genome.Recently, we generated a synthetic baculovirus Ac MNPV-WIV-Syn1 which fully resembled its parental virus Autographa californica multiple nucleopolyhedrovirus(Ac MNPV). Here, we report the modification of Ac MNPV-WIV-Syn1 into a novel bacmid, Ac Bac-Syn, which can be used as a backbone for Bac-to-Bac system. To achieve this, a vector contained a Lac Z:att Tn7 and egfp cassette was constructed, and recombined with a linearized Ac MNPV-WIV-Syn1 genome by transformation-associated recombination in yeast to generate bacmid Ac Bac-Syn. The bacmid was then transfected to insect cells and the rescued virus showed similar biological characteristics to the wild-type virus in terms of the kinetics of budded virus production, the morphology of occlusion bodies, and the oral infectivity in insect larvae. For demonstration, a red fluorescent protein gene Dsred was transposed into the att Tn7 site by conventional Bac-to-Bac method, and the transfection and infection assays showed that Ac Bac-Syn can be readily used for foreign gene insertion and expression.Ac Bac-Syn has several advantages over the conventional Ac MNPV bacmids, such as it contains an egfp reporter gene which facilitates visualization of virus propagation and titration;its DNA copy numbers could be induced to a higher level in E. coli;and the retaining of the native polyhedrin gene in the genome making it an attractive system for studying the functions of gene related to occlusion body assembly and oral infection.