Abstract
Recent technical advances on assembling DNA fragments has enabled generation of large, circular DNA of which length reached several hundred-kilobases. Adaptation of such technologies to baculoviral genomics would allow for flexible manipulation of baculoviral genomes and thus generation of designed viral genomes for both basic and applied biology. Here we report that a BmNPV genome-size DNA could be assembled from a set of PCR-amplified DNA fragments and was infectious to Bombyx mori ovary-derived BmN cell line. The DNA fragments were designed (1) to be excised mainly at intergenic regions and (2) with no homologous sequences on the terminals among the fragments for assembly purpose, and (3) with their lengths and Tm values of priming sequences adjusted for simultaneous amplification. Although these fragments were initially designed for assembling with Gibson assembly and in yeast in the first and subsequent steps, respectively, the bacmid propagated in yeast during a feasibility test showed a deletion during in-yeast assembly and/or propagation. Therefore, we assembled the fragments using only Gibson assembly throughout the assembling steps. An assembled clone was chosen and confirmed to carry the inverted vector sequence, a watermark to distinguish the assembled clone from the parental clone, and to be infectious to BmN upon transfection. The design and assembly strategy of DNA fragments and both amplified and assembled fragments become the foundation for further development of BmNPV-based, designed genomes.
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