Relative levels of c-di-GMP were estimated using the c-di-GMP biosensor plasmid pFY4535 (Zamorano-Sanchez et al., 2019). This plasmid contains the gene for red fluorescent protein (RFP) under the control of a c-di-GMP-binding riboswitch. It also contains the gene for AmCyan protein, expressed independently of c-di-GMP. Increasing levels of c-di-GMP result in increased production of RFP and thus increased ratios of RFP to AmCyan. For liquid measurements, strains carrying pFY4535 were grown in Gent-containing LBS medium with shaking overnight at 28°C and then subcultured for 16–24 h under the same conditions. Liquid cultures were concentrated by centrifugation and resuspended in 1 ml of phosphate-buffered saline (PBS). The samples were then washed three times with PBS, and 1 μl of the washed samples was added to 1 ml of PBS. These diluted samples were evaluated for production of RFP and green fluorescent protein (GFP) using the LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, United States) using AmCyan and PE-TexasRed channels, and the data were analyzed via FlowJo software (Ashland, OR, United States). The resulting data were first gated on live cells and then for AmCyan and RFP double-positive cells. The resulting populations were depicted using a representative histogram of PE-TexasRed (RFP) levels to highlight relative differences in c-di-GMP. The y-axis was normalized to mode to account for differences in event count of the samples. The geometric mean fluorescence intensities of the PE-TexasRed curves were quantified and analyzed in the accompanying graphs.

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