Moflo astrio eq cell sorter

Cells containing enriched protease sequences as determined by DNA sequencing (incidence > 1) were picked and cultured as described already. GFP fluorescence intensities of the cells were analyzed using a Gallios™ flow cytometer (Beckman Coulter). Cells containing the top five protease candidates based on GFP fluorescence signals were cultures as described and GFP and mCherry fluorescence intensities were analyzed using a MoFlo Astrio EQ cell sorter (Beckman Coulter).

Approximately 3.0 × 10 9 cells co-expressing the plasmid encoding random mutagenesis 3C proteases and the reporter plasmid encoding the 3C substrate LEVLFQ↓GP were pelleted (1500 g, 4°C, 4 min). The pellet was washed in phosphate buffered saline (PBS) before resuspension in 1 ml PBS. The cells were sorted based on GFP and mCherry signals. Three rounds of cell sorting were carried out with increasing sorting stringency for GFP and mCherry signals (1.5%, 0.5% and 0.1% of cells within the sort gate, respectively). A total of 5.0 × 10 8 cells were sorted in the first round to oversample the number of transformants 20-fold. In subsequent rounds a 10-fold excess of previously sorted cells was sampled. The cells were sorted using a MoFlo Astrio EQ cell sorter (Beckman Coulter). In the last round, twice 100 cells were sorted directly onto two LB agar plates containing appropriate antibiotics (100 cells per plate), and incubated at 37°C for 16 h. Ninety-six single colonies were picked and the sequences of the protease plasmids were analyzed by DNA sequencing (Microsynth).