Sapphire lp laser

All chemicals used for media and other experiments are from Sigma (St. Louis, MO) unless otherwise noted. Synechocystis 6803 cultures of WT, CB, CK, and PAL were initially inoculated into 20 ml of liquid TES-buffered BG11 medium [19 ] and grown for 7 days in a Sanyo Versatile Environmental Test Chamber at 30°C under 20 μmol photons m^-2 s^-1 white fluorescent light with constant shaking at 125 rpm. Due to the different growth rates among the strains, cultures were sub-cultured based on cell concentration. The concentration of Synechocystis 6803 cultures were determined by flow cytometry using a BD Influx Fluorescence Activated Cell Sorter (FACS, BD Biosciences, San Jose, CA). Upon harvesting, 100 ml of each sample was analyzed using the 488-nm laser excitation from a Sapphire LP laser (Coherent Inc., Santa Clara, CA) at 100 mW. Forward and side scatter were used to gate out cellular debris and values are recorded. Optimization and calibration of the FACS was performed before each analysis using 3 μm Ultra Rainbow Fluorescent Particles (Spherotech, Lake Forest, IL). Cultures were then grown for 5 days, and harvested by centrifugation for proteomics analysis. Growth medium was supplemented with antibiotics as follows: CB and CK, 10 μg/ml kanamycin; PAL, 10 μg/ml chloramphenicol and spectinomycin [20 (link)].

The flow cytometry data were obtained using a BD Influx fluorescence-activated cell sorter (FACS; BD Biosciences, San Jose, CA). Using the 488-nm excitation from a Sapphire LP laser (Coherent Inc., Santa Clara, CA) at 100 mW, samples were analyzed using a 70-μm nozzle. Optimization and calibration of the FACS were performed before each analysis using 3-μm Ultra Rainbow fluorescent particles (Spherotech, Lake Forest, IL). The ratio of the two distinct populations of cells within a mixed microbial community was identified from 50,000 recorded cells using size and complexity gates with FCS Express (Los Angeles, CA) flow cytometry software. Cell counts are presented as the percentage of total counting events.