Journal: Viruses

Article Title: A Baculovirus Expression Vector Derived Entirely from Non-Templated, Chemically Synthesized DNA Parts

doi: 10.3390/v15101981

Figure Lengend Snippet: SynBac1 genome is replication-competent but exhibits a 10-fold replication defect relative to bMON14272. bMON14272 and SynBac1 bacmid DNA was isolated from two independent E. coli clones. Each preparation of bacmid DNA was transfected into Sf9 insect cell culture, and the kinetics of the budded virus output were quantitated via TCID50 assay. Error bars depict standard error of the mean between duplicate transfected cultures.

Article Snippet: The PCR product was combined in Gibson assembly with MluI-digested bGGVoy and transformed into NEB ® 10-beta E. coli .

Techniques: Isolation, Clone Assay, Transfection, Cell Culture, Virus, TCID50 Assay

Journal: Viruses

Article Title: A Baculovirus Expression Vector Derived Entirely from Non-Templated, Chemically Synthesized DNA Parts

doi: 10.3390/v15101981

Figure Lengend Snippet: SynBac2 genome replicates in Sf9 cells comparably to bMON14272. ( A ) The respective B subfragments encoding the lysine quadruplet in LEF8 and the proline triplet in P49 were modified to match the amino acid sequence of these genes in bMON14272 and our sequence-confirmed wild-type Ac MNPV E2 sample. These new B subfragments were used to assemble new A regions and a second synthetic baculovirus genome known as SynBac2. ( B ) Bacmid preps isolated from three independent E. coli clones were transfected into Sf9 cells, and the resulting budded virus was quantitated via TCID50 assay. Error bars indicate standard deviation between cultures transfected by the three bacmid preps for each design. ( C ) To obtain single-cycle replication kinetics, the budded virus-containing supernatant from ( B ) was used to infect new Sf9 cultures at a multiplicity of infection of 3 TCID50 per cell. The resulting budded virus output was quantitated via TCID50 assay. Error bars indicate standard deviation between cultures infected by the triplicate baculovirus stocks obtained from the three independent bacmid preps.

Article Snippet: The PCR product was combined in Gibson assembly with MluI-digested bGGVoy and transformed into NEB ® 10-beta E. coli .

Techniques: Modification, Sequencing, Isolation, Clone Assay, Transfection, Virus, TCID50 Assay, Standard Deviation, Infection