- Five different mutant strains were selected for investigation, based on their growth data and theoretical prediction about potassium sequestration capability. (See mutants information)
- Six different concentrations of potassium were prepared by adding 1ml of 10x KCl stock solution to 9ml NOK broth, to make final [K+] of 0, 1, 5, 10, 50 & 200mM.
- Each mutant was innoculated into 10ml NOK samples of different potassium concentrations, and grown overnight at 37°C in a shaking incubator.
- In the morning, OD600 recorded in 1ml cuvette and converted into cell number using calibration graph. Cells were kept on ice at all times after this.
- 500ul of this culture was extracted & centrifuged (5 min, low speed.)
- Supernatant removed and discarded (no further analysis due to viscosity problems)
- Pellet washed (resuspend in 1ml SDW and centrifuge 5 min, low speed.)
- Pellet resuspended in 1.5ml SDW, cells then fractured by 3 rounds of freeze-thaw lysis in liquid nitrogen.
- Flame photometry performed on most sensitive setting (See calibration graph)
- This was converted into data below using our 'Potassay'(potassium assay) spreadsheet.
- Four repeats were performed during the course of the day.
Note: small horizontal markers above and below points on graphs shown indicate range of data (results from 4 repeats)