Figure 1

Bipartite selection provides significant increase in selection efficiency for lambda red Bacmid recombination. A) Two recombination cassettes were designed, both with baculovirus flanking sequences (AcMNPV), p35 promoter (P_p35), Renilla Luciferase (R-luc) and polyhedrin polyadenylation sequences (T_ph) and LoxP sites flanking a bacterial selectable marker. For bipartite selection (top) a Zeocin resistance gene (ZeoR) and LacZα marker were incorporated. For single marker selection (bottom) the cat gene, conferring chloramphenicol resistance, was incorporated. B) The number of positive colonies following red recombination using 30 ng of each recombination cassette (~12.58 fmol), generated either by PCR amplification or restriction enzyme digestion, and BACmid DNA. Each transformation was carried out in triplicate, error bars indicate the standard deviation of the results. The data for the two different selections were clustered and a t-test used to confirm that the results were statistically significant (p = 0.03). C) PCR confirming recombination and correct target site 12 independent bacmid recombinants (lanes 1-12). One primer annealed to genomic DNA flanking the target insertion site and the other to sequence inside the recombination cassette. Only recombination at the correct genetic locus would produce the PCR product (arrowed). Lane 13, no template PCR reaction; Lane 14, bMON14272 Bacmid template DNA; Lane 15 plasmid containing the entire DNA used for recombination as template. D) Renilla luciferase activity at 48 hours post infection in cell lysates from cells infected with passage 2 of recombinant bacmids 1-12 from B. Cells and AcMNPV indicate background activity in lysates from uninfected and unmodified bacmid infected cells, respectively.