Mutants

Mutant strains were required to identify and remove any interference from E. Coli's natural K+ homeostatic mechanisms. The following mutants were ordered from [http://cgsc.biology.yale.edu| 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.

Checking Competence

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.

Growth curves

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, compared to the calibration. Their final K+ concentrations were also measured after 24 hours growth.