Part:BBa_K1968021:Experience

Applications of BBa_K1968021

Promoter Sca6-2 was characterized in E. coli TOP10 cells. Colonies were picked and grown overnight at 37 °C, 250 RPM in 50 mL conical tubes in 10 mL of LB medium with its respective antibiotic (50 ug/mL kanamycin). Cell density was measured in a Jenway 7305 spectrophotometer, the obtained data was processed using Excel Data Processing tools. Cells were then subcultured into opaque-walled 96 well plates and adjusted to OD600 0.05 in 290 µl of LB medium supplemented with antibiotics as well as their respective inducer concentration (0, 0.005, 0.01, 0.05, 0.1 and 0.5 mM iPTG) in triplicates. Plates were then placed in the Fluostar omega microplate reader from BMG labtech, and grown at 37 °C for 20 hours at 300 RPM. Aside from monitoring OD600, fluorescence was determined by measuring emission wavelengths using two fixed excitation filters (584 and 544 nm) and two fixed emission filters (590 and 512 nm) every 30 minutes. Triplicate replicates were performed for each inducer concentration. All data is presented in relative GFP fluorescence units, which represents the relative amount of the specific wavelengths of light collected by the plate reader at the conditions described above.

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