Fig. 1.

The MultiBac baculovirus expression vector system. MultiBac consists of a baculoviral genome that we engineered for optimal multigene delivery and protein complex expression (left). The MultiBac genome is propagated as a bacterial artificial chromosome (BAC) in Escherichia coli cells that supply the Tn7 transposition function from a co-existing helper plasmid. Several methods have been successfully applied to assemble expression cassettes containing genes of interest and gene regulatory elements. We introduced tandem recombineering (TR), a method that relies on iterative cycles of sequence and ligation independent cloning (SLIC) coupled to Cre-mediated fusion of plasmid DNA precursor elements [9, 19]. Other alternatives include the uracil-specific excision reagent (USER) and biGBac methods [20, 21], in addition to conventional restriction/ligation-based cloning techniques. We engineered a second entry option into the viral backbone, which can accept additional functionalities by site specific recombination into the viral LoxP site. Composite MultiBac baculoviral DNA containing all DNA elements of interest is extracted. Transfection of insect cell cultures in small scale yields live MultiBac virions. These can be used for a wide range of applications (right). The viral LoxP site is shown as a circle filled in red. ORF open reading frame, SBDD structure-based drug design, HCS high-content screening, VLP virus-like particle, Cryo-EM structure determination by electron cryo-microscopy, X-ray X-ray crystallography, Kan kanamycin resistance marker, Amp ampicillin, LacZ LacZα gene enabling blue/white selection, mini-attTn7 attachment site for Tn7 transposition. The schematic drawing of the baculovirion was kindly provided by Kari Airenne