Fig. 5

Phenotypic results from high-throughput conversion of GFP strains to RFP-G418 strains. Targeting results are indicated here for 68 strains in which a GFP signal was visible in the starting strain. Trial A and Trial B are distinguished by the separate methods indicated in Fig. 4b. The conversion from GFP to RFP is indicated in all columns by the red color. Trial A indicates microscopy results for all strains except the three indicted in black which did not form a colony on G418. Strains indicated in grey we were unable to unequivocally screen using microscopy and the remainder showed a phenotype consistent with RFP, GFP or a mixed population of both, as specified. Further analysis was then undertaken on some of the strains in Trial A, where secondary microscopy checks were performed on cells from a mixed-population colony and from a single clonal colony. The clonal colonies were then also checked for insertion of the template DNA by PCR, results indicate the detected genotype. Secondary testing for growth on 5-FOA plates shows two strains with a URA+ phenotype, indicating that the template plasmid has not been effectively counter-selected against. The methodology for Trial B was adapted to counteract this. In Trial B, cells from a mixed colony were checked with microscopy as in Trial A, then cells from mixed populations were checked again. Where possible, 3 clonal colonies were then assessed by fluorescence microscopy and PCR. The observations from each of the three colonies are indicated