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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.

• 1st of July:

B. subtilis EtOH resistance assay was continued in a narrow concentration range.

E. coli (DH5alpha) cells were transformed using BioBricks required for EtOH production.

O/N cultures of E. coli and B. subtilis were set up to continue with the experiments tomorrow.

• 2nd of July:

Transformation was successful. Four colonies were selected and propagated into 5ml LB bottles supplemented with adequate antibiotic.

E. coli cells plated for competitors were harvested and propagated into 50 ml LB medium. Following 4h incubation they were used to make competent cells for future transformations.

B. subtilis resistance to heavy metals experiment was continued in a narrow metal ion concentration range.

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 diluting the culture with high volumes. 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.

• Transformation of competent E. coli cells':

1.Use 100ul aliquots of competent E. coli cells from the -80 deg. C freezer.

2.Place cells directly on ice. Try to limit the exposure to heat (i.e. do not touch the bottom of the eppendorf)

3.Add 2 ul of plasmid DNA to the cells and incubate on ice for 30 - 60 minutes.

4.Heat shock the cells in 42 deg. C for 90 seconds (exact timing)

5.After heat shock place the cells on ice for additional 90 seconds.

6.Add 900 ul of LB medium to the cells.

7.Grow in 37 deg. C shaking for 45 - 60 minutes.

8.Plate 100 ul of cells on a plate with appropriate antibiotic.