Different efficiencies of incorporation into the polyhedra crystals obtained with the different fragments studied
. A, flow cytometry studies of purified polyhedra crystals obtained with the different fragments indicated in the figure. In all cases a MOI of 1 was used for each EGFP containing polyhedrin fragment and a MOI of 3 for wild type polyhedrin. Crystals purified from Sf9 cells subjected to sonication (Methods). Fluorescence intensity collected in the 525 nm emission channel (Methods). In all cases 10,000 events were collected for each polyhedra. Autofluorescence (fluorescence background) was determined using wild type polyhedra (without EGFP), as indicated in the first panel at the top. Using this background level we identified the EGFP positive fluorescence (EGFP+, indicated by the gray rectangle). B, percentage of EGFP+ events (individual polyhedra crystals) obtained from the histograms shown in A. Notice polyhedra crystals produced with fragment PH(58–110)EGFP produced the highest EGFP intensity values, followed by PH(1–58)EGFP and PH(1–110)EGFP. Notice that PH(1–25)EGFP, PH(25–58)EGFP and PH(110–245)EGFP did not produce fluorescent polyhedra. Flow cytometry data is in agreement with the results obtained with confocal microscopy (Figure 7).