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From 2013.igem.org

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Chronological notebook

• 27th of June:

B. subtilis (strain: 168) culture inoculated into 10 ml LB medium. Overnight incubation in 37 deg.C

Preparation of metal ion stock solutions: Ni - 0.1M; Zn - 0.1M; Fe - 0.5M; Cu - 0.5M; Co - 0.1M; Mn - 1M

• 28th of June:

0.5 ml units of O/N culture diluted to 50ml (x100 dilution in LB medium). 3 ml of diluted culture aliquots poured into universal bottles (enables adequate aeration of the culture). Those were inoculated with 30 ul of differently concentrated metal ion solutions giving final concentrations of:

Ni - 1mM; 0.75 mM; 0.5 mM and 0.25mM; Zn - 1mM; 0.75 mM; 0.5 mM and 0.25mM; Fe - 5mM; 3.75mM; 2.5mM and 1.25mM; Cu - 5mM; 3.75mM; 2.5mM and 1.25mM; Co - 1mM; 0.75 mM; 0.5 mM and 0.25mM; Mn - 10mM; 7.5mM; 5mM; 2.5mM; 1.25mM and 0.625mM

0.4 ml of O/N culture diluted to 40ml (x100 dilution in 2xLB medium). 3 ml of diluted culture aliquots poured into universal bottles (enables adequate aeration of the culture). Those were inoculated with 1ml of differently concentrated ethanol solutions giving final concentrations of:

2%; 4%; 6%; 8%; 10%; 15%; 20% and 25%

The initial Absorbance readings at OD600 for diluted cultures were taken. Following 7h. incubation in 37 deg.C absorbance of each sample was measured and compared to samples inoculated only with H2O.

Recipes (or protocols if you prefer so)

• 2xLB medium:

Tryptone, 20g Yeast extract, 10g NaCl, 10g (when making more medium add 20g) Dissolve in 800 ml dH2O, adjust pH to 7.0 with NaOH/HCl, adjust volume to 1 litre and autoclave.

NOTE: use this medium when dilluting the culture with high voulmes. If possible try to use same volume of 2xLB and the solution you want to add to it as B. subtilis does not grow really well in highly concentrated LB.