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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 (Aleksandra, Harry and Jan)

• 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 (Kyle, Harry, Aleksandra, Dainius and Jan).

• 1st of July:

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

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

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

• 2nd of July:

Transformation was successful. Four colonies were selected and propagated into 5ml LB bottles supplemented with adequate antibiotic (Jan). After 8h incubation in 37 deg. C cells were pelleted and plasmids were extracted using Qiagen MiniPrep Kit (Aleksandra). Concentration and quality of DNA was measured using NanoDrop.

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 (Aleksandra and Jan).

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

• 3rd of July:

Since we propose using B. subtilis as a chassis for our waste water treatment bacteria, we first set about determining the concentration of different metal ions that the non genetically altered chassis can cope with. We initially observed cell density of cultures after 8 hours incubation with varying metal concentrations. Following this we recorded culture density every hour to obtain growth data for appropriate concentrations of metals, as indicated by the initial 8 hour cultures. These experiments continue... (Harry, Danius, Hugo)

• 4th of July:

The competent E. coli cells were tested by transforming them with parts for ethanol production. The negative control was performed to confirm that cells have no indigenous antibiotic resistance and that prepared antibiotic plates have antibacterial properties (Aleksandra and Jan).

Time-lapse measurements of B. subtilis growth in specified metal concentrations (based on previous data) were performed (Harry, Hugo, Dainius and Kyle)

Team started preparing poster (Kyle) and presentation (Dainius and Hugo) for the Young Synthetic Biologist meeting held in London over the next weekend: 12-14/07/2013

• 5th of July:

Outcome of the transformation was positive with the negative control showing no growth (as expected). Four colonies were collected and grown in 5ml LB supplemented with adequate antibiotic. Following 7h incubation the entire culture was pelleted and plasmid was purified using Qiagen MiniPrep Kit. NanoDrop was used to measure the DNA concentration (Harry and Jan)

Time-lapse measurements of B. subtilis growth in specified metal and ethanol concentrations (based on previous data)were performed (Harry, Hugo, Dainius and Jan).

• 8th of July:

Two PCR reactions were set up to clone out genes of interest from B. subtilis (Harry and Hugo; Dainius and Kyle).

The ethanol production part was combined with a promoter - overnight ligation(Jan)

• 9th of July:

The PCR product was run on the gel to confirm the presence of the desired product (Aleksandra, Dainius, Jan)

The ligation product was transformed into the competent E. coli cells. Those were plated on the KAN+IPTG+X-gal plates (Aleksandra, Jan).

B. subtilis genomic DNA was purified from the O/N culture for the use in the future PCR reaction (Harry)

• 10th of July:

DNA gel electrophoresis of the PCR product was performed once more due to inconclusive results from 9/07/2013 (Jan). Bands of expected size were observed. Additional PCR was set up to obtain more DNA (Harry and Hugo). Following gel electrophoresis resulting products were restricted using correct enzyme and an O/N ligation reaction was set-up (Aleksandra and Jan)

The transformation using the EdiBrick and ethanol production parts ligation product was successful - numerous blue colonies were obtained. Two colonies were selected and propagated into 5ml LB +KAN medium. Two more colonies (4 in total) were propagated onto LB+KAN+X-gal plates and incubated O/N in 37 deg.C (Aleksandra, Jan)

• 11th of July:

The ligation products of vector and two transcription factors amplified using PCR were transformed into E. coli and plated on LB+AMP+X-gal+IPTG plates (Harry and Weike)

• 15th of July:

The transformation done using the ligation product gave negative results. The restriction of SinR; Fur and AmpR vector was repeated. O/N ligation was set up using new restriction products (Harry, Weike and Jan)

Cells from four different plates of the EdiBrick_pET ligation were resuspended in 5 ml LB medium + KAN and grown in 37 deg.C. The plasmid was purified following 4 hours of incubation using Qiagen MiniPrep Kit (Aleksandra and Jan)

• 16th of July:

The DNA resulting from ligation was used for transformation of competent E. coli. Cells were plated on LB+AMP+IPTG (20 ul) plates (Weike and Hugo)

The EdiBrick_pET DNA was restricted using XbaI and PstI in CutSmart buffer. 2 h. incubation in 37 deg. C was used for restriction reaction.The resulting proucts were run on the gel (15ul product + 3ul loading dye). Three out of four DNA samples showed correct band sizes (Aleksandra).

Reagents required for ethanol assay were prepared and a sample data for 0; 0.5 mM and 1 mM were obtained in order to plot a standard curve Aleksandra and Jan).

Timelapse analysis of B. subtilis growth in varying concentrations of three different heavy metals was performed (Harry and Dainius)

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.

• Preparation of competent E. coli cells:

TSS buffer is required for this process. 24 ml TSS:

-17 ml LB -5 ml 40% w/v PEG 3350 -1 ml 1 M MgCl2 filter sterylise the mix above and add: -1 ml DMSO

Procedure:

Propagate E. coli from plate into 50 ml of LB medium. Grow until OD600 of about 0.2 to 0.5 is obtained. Pellet 1 ml of culture for each eppendorf of competent cells. Remove supernatant and add 100 ul of ice-cold TSS buffer. Incubate with TSS for 30 min on ice and store in -80 deg.C

• 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.