Computational protocol: In vivo selection of sfGFP variants with improved and reliable functionality in industrially important thermophilic bacteria

Similar protocols

Protocol publication

[…] Single-cell fluorescence measurements were made with a FACSCanto flow cytometer (BD Biosciences) with 488 nm excitation from a 20 mW solid-state laser. Geobacillus pre-cultures were obtained by inoculating 10 mL TGP supplemented with 8 μg/mL chloramphenicol from glycerol stocks and incubation at 60 °C and 170 rpm. After 16 h, cells were diluted to an OD600 of 0.08 into 25 mL pre-warmed, fresh TGP-Cm8 in 250-mL baffled flasks, and incubated at the desired temperatures at 170 rpm. Samples taken after specific time intervals (1, 2, 4, 6, 8, 10 and 12 h) or at an OD600 of 1.0 were immediately diluted (at least 1:100) with pre-warmed phosphate buffer solution (PBS, pH 7.0). Per sample, 100,000 events were recorded by measuring the fluorescence at 530 nm/30 nm band-pass width. The instrument settings were as follows: threshold-SSC 200, FCS 200, FL1 600 V, all in log amplification. The data were captured with the BD FACSdiva software (version 5.0.3) and for data analysis Flowing Software (version 2.5.1) and FCSalyzer (version 0.9.13) were used. […]

Pipeline specifications

Software tools BD FACSDiva, Flowing Software
Application Flow cytometry
Organisms Escherichia coli
Chemicals Amino Acids, Nucleotides