Method

• 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.

Results

Potassay raw data & calculations in Excel spreadsheet

Note: small horizontal markers above and below points on graphs shown indicate range of data (results from 4 repeats)

BW25113 Graphical Analysis (Control)	JW3314 Graphical Analysis (Kef G-)	BW30166 Graphical Analysis (Kdp E-F-)	JW0045 Graphical Analysis (Kef F-)
JW1242 Graphical Analysis (Kch-)