Figure 2

Work-flow diagram used for the reverse genetics approach. A . Generation and selection of transformants. Thousands of transformants are generated using the AphVIII marker gene and selected on paromomycin-containing plates. B . Isolation of individual transformants. Individual colonies are cultured in 96-well microtiter plates (200 μl per well). C . Transformant pooling. Aliquots (25-50 μl) from each well of an individual 96 well microtiter plate are pooled and cultured in fresh medium (pool). D . Isolation of genomic DNA from pools. DNA from each pool of transformants is isolated and diluted to 100 ng/μL. E . Generation of DNA superpools. Superpools are constructed by combining equal volumes of genomic DNA from 10 different pools. F . Screening the superpools. A set of independent PCR reactions using marker and target gene primers is performed with each DNA superpool as template (in the real case depicted, amplification occurred in the superpool sample loaded in lane 16) G . Confirmation of PCR products. Amplified PCR products are sequenced using the marker gene primer. H . Screening specific pools. The 10 different pools of DNA that comprise the superpool are individually screened using the appropriate primer pair. I . Screening of individual transformants. Positive transformants are identified within a specific microtiter plate by colony PCR. J . Isolation of specific transformants. Cells from the well containing the positive transformant are streaked onto solid medium to obtain single cell-derived colonies, which are then screened by colony PCR. This step is used to eliminate potential cross-contamination among transformants.