Mutant strains were required to identify and remove any interference from E. Coli's natural K+ homeostatic mechanisms. The following mutants were ordered from CGSC.
Strain Code Mutation Resistance BW25113 Control - BW27564 Kdp E-F - BW30166 Kdp E-F - JW0045 Kef F Kan JW0683 Kdp D Kan JW1242 Kch Kan JW3314 Kef G Kan JW5096 Kdp E Kan TK3D11 Kdp -
These strains were then plated out. The Kanamycin resistant strains were streaked onto kanamycin plates (50μg/mL) and also onto non-antibiotic plates. The non-resistant strains were plated onto plain plates. After growing overnight at 37°C single colonies were picked into 15μL broth (containing kanamycin if appropriate), and also streaked onto fresh plates. After growing overnight at 37°C glycerol stocks were made in triplicate and frozen at -20°C. The rest of the broth was stored at 4°C, as were the plates.
To check all the mutants could be made competent they were transformed with pUC19 and grown on ampicillin plates. Three strains (BW30166, JW142 and TOP10) were also transformed with Lac inducible YFP (BBa_I5311), and grown on both inducer and repressor plates.
All the mutant strains were grown in 3 concentrations of K+ in LB - 1mg/mL, 50mg/mL and 200mg/mL - and their growth measured using the OD600. Their final K+ concentrations were also measured after 24 hours growth.