Fig. 1.
PclpB–lacZ fusion reports on the presence of the Sup35 NM prion in E. coli cells. (A) Colonies formed by an experimental sample starter culture of PclpB–lacZ reporter strain cells containing Sup35 NM and New1 fusion proteins. After overnight growth at the permissive temperature, the starter culture cells were plated on indicator medium and grown at a temperature nonpermissive for pSC101TS-NEW1 replication. Colonies were photographed after ∼24 h of growth. The white arrow indicates a dark blue colony among pale blue colonies. (B) Dark blue colony counts, total colony forming units (cfu), and the corresponding dark blue colony frequencies (percentage dark blue) are reported for colonies generated from plating the indicated starter cultures. Starter cultures contained the Sup35 NM plasmid and either the New1 temperature sensitive (t.s.) plasmid (+) or the corresponding empty vector (−). The percentage values reflect the sample-size weighted mean of six independent experiments. The SEM of all experiments is reported for each starter culture type. (C) SDS-stable Sup35 NM aggregates are detected by a filter retention assay in cell extracts from 10 of 10 cultures inoculated with dark blue colonies (Top) and 0 of 10 cultures inoculated with pale colonies (Bottom). All colonies were derived from the Sup35 NM + New1 experimental sample starter culture. For each sample, cell extracts were serially diluted threefold. The experimental sample starter culture (Sup35 NM + New1) serves as the positive control (+), whereas the control sample starter culture (Sup35 NM + empty vector) serves as the negative control (−). The α-His antibody recognizes the Sup35 NM-mYFP-His6x fusion protein. See also SI Appendix, Fig. S1. This analysis was performed for two additional experiments (including a total of 24 dark blue colonies and 24 pale blue colonies), and the same correlation between colony color phenotype and the presence or absence of aggregates was observed.