IGEM:Cambridge/2008/Notebook/Voltage/K+ Concentrations: Difference between revisions
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=Method= | =Method= | ||
*Five different mutant strains were selected for investigation, based on their growth data and theoretical prediction about potassium sequestration capability. [[IGEM:Cambridge/2008/Voltage/Mutant Strains |(See mutants information)]] | |||
*Five different mutant strains were selected for investigation, based on their growth data and theoretical prediction about potassium sequestration capability. [[IGEM:Cambridge/2008/Notebook/Voltage/Mutant Strains |(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. | *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. | *Each mutant was innoculated into 10ml NOK samples of different potassium concentrations, and grown overnight at 37°C in a shaking incubator. | ||
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[[Media:MutantPotassium.xls |Potassay raw data & calculations in Excel spreadsheet]] | [[Media:MutantPotassium.xls |Potassay raw data & calculations in Excel spreadsheet]] | ||
[[Image:Kallstrains.JPG | 400px |left]] | |||
[[Image:Kallstrains2.JPG | 400px |right]] | |||
Note: small horizontal markers above and below points on graphs shown indicate range of data (results from 4 repeats) | |||
<gallery> | |||
Image:KBW25113.JPG|BW25113 Graphical Analysis (Control) | |||
Image:KJW3314.JPG|JW3314 Graphical Analysis (Kef G-) | |||
Image:KB30166.JPG|BW30166 Graphical Analysis (Kdp E-F-) | |||
Image:KJW0045.JPG|JW0045 Graphical Analysis (Kef F-) | |||
Image:KJW1242.JPG|JW1242 Graphical Analysis (Kch-) | |||
</gallery> |
Latest revision as of 08:16, 3 September 2008
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-)