- Dissolution of TPA in DMSO (Dimethyl sulfoxide). The best concentration obtained is 62 mg/l
- Dilution of TPA stock in LB Broth => TPA precipitates at a concentration of 3.9 g/l, maximum concentration in LB Broth is 0.9 mg/l
Preparation of M9 and minimal media
3 solutions:
Solution 1: (salts)
5.3 g of Potassium Phosphate (dibasic) K2HPO4
2 g of Potassium Phosphate (monobasic) KH2PO4
1 g of Ammonium Sulfate (NH4)2SO4.
0.5 g of Sodium Citrate (tribasic, dihydrate)
Autoclave
Complete with water to 333 ml
Solution 2: (agar)
16 g of agar
Complete with water to 333 ml
Autoclave
Solution 3: (sugar)
In fact we have to put 4 g of sugar in 333 ml of H2O. We wanted to see if E. coli bacteria took TPA as a carbon source. So just the water was added. Next, the flask was autoclaved.
When the three parts have been autoclaved, 2 more ingredients must be added to the medium aseptically:
1 ml of 10% magnesium sulfate MgSO4 (autoclaved too) (1.5 g in 15 ml of H2O).
1 ml of 0.2% Thiamin (vitamin B1).
At the end the volume of Agar was exceeded (350 ml and not 333 ml). So we had to redo this experiment the next day.
05-08-15 – Experiment: Preparation of M9 medium – VB, PV
Redaction by VB
Aim: Get culture medium for BAP 1 Pfeifer cells.
Protocol: (for 1 l medium)
Agar was directly put in a bottle and salts were dissolved in a beaker with water.
6 g of Sodium Phosphate Na2HPO4.H2O
3 g of Potassium Phosphate KH2PO4
0.5 g of Sodium chloride NaCl
1 g of Ammonium chloride NH4Cl
16 g of agar
Complete with water to 1l
When this flask was autoclaved some ingredients were added in aseptical conditions:
1 ml of 1M Magnesium sulphate MgSO4 (6.02 g in 50 ml H2O)
1 ml of 0.1 M of Calcium Chloride CaCl2 (0.55 g in 50 ml of H2O)
A mistake was done yesterday but this time an other did too: The last ingredients were added but they were not sterile so the medium failed again.
06-08-2015 – Experiment: Minimal and M9 medium for TPA Toxicity and BAP 1 – VB, PV
Redaction by VB
Aim: Have prepared M9 medium and minimal medium for Petri dishes
We restarted the last two protocols but this time we included the agar for the future Petri dishes.
For the future experiments every medium will have the same concentration of sugar for the bacteria growth, and the test of the TPA toxicity. We began by a theoretical concentration for our Petri dishes: to see the saturation concentration of TPA we decided to do dilutions at 1/10 and 1/20.
This time we decided to make 1.5 l of minimal medium
Protocol:
Solution 1: (salts)
8 g of Potassium Phosphate K2HPO4
3 g of Potassium Phosphate KH2PO4
1.5 g of Ammonium Sulfate (NH4)2SO4
0.75 g of Sodium Citrate (tribasic, dihydrate) Na3C6H5O7.(H2O) 2
Complete with water to 1 l
Solution 2: (agar)
24g of Agar
Complete with water to 500 ml
Solution 3:
500 ml of H2O
We underestimated the time for agar to solidify, so we had solid agar in the test-tube. Moreover one of the flask was not sterile so we contaminated our medium. We decided to return at the first protocol (for 1l).
At the end of this journey the two media were ready but they did not have the last ingredients.
Enzymatic Assay of NB-Esterase
- XbaI ans PstI enzymatic digest of the Biobrick BBa_K808030 and pDG011 plasmid.
- Gel purification of the digested fragments on 0.8% agarose gel.
- Ligation of pDG011 plasmid and BBa_K808030 using T4 DNA ligase.
- Gel migration on 0.8% agarose gel.
Figure 8: Gel for verification of ligation.
There is no band for the ligation sample.
Gibson Assembly
- Ligation of ThpA1, Transp_, 1392932_, using T4 DNA ligase.
- DNA concentration measurement using a spectrophotometer.
Biobrick |
DNA Concentration (ng/µl) |
OD (230 nm) |
OD (260 nm) |
OD (280 nm) |
OD (260 nm) / OD (280 nm) |
OD (260 nm) / OD (230 nm) |
808010 |
30.00 |
9.33 |
0.75 |
0,30 |
1.87 |
0.06 |
808011 |
50.00 |
11.78 |
1.00 |
0.63 |
1.61 |
0.09 |
808012 |
17.50 |
10.93 |
0.33 |
0.18 |
0.84 |
0.03 |
808013 |
5.00 |
10.55 |
0.10 |
0.06 |
1.56 |
0.01 |
80801v |
22.5 |
12.13 |
0.47 |
0.30 |
1.54 |
0.04 |
1095000 |
37.50 |
18.48 |
0.78 |
0.43 |
1.83 |
0.04 |