Week 10

08/03 - 08/07

Toxicity and solubility of TPA (Terephthalic acid)

1. Dissolution of TPA in DMSO (best concentration of 62 mg/l)
2. 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 – 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



1. XbaI ans PstI enzymatic digest of BBa_K808030 and pDG011
2. Gel purification of the digested fragments on 0.8% agarose gel.
3. Ligation of pDG011 and BBa_K808030 using T4 DNA ligase.
4. Gel migration on 0.8% agarose gel.

Figure 8: Gel for verification of ligation.
There is no band for the ligation sample.

Gibson Assembly

5. Ligation of ThpA1, Transp_, 1392932_, using T4 DNA ligase.
6. DNA concentration measurement using a spectrophotometer.

Biobrick	DNA Concentration (ng/µL)	OD (230nm)	OD (260nm)	OD (280nm)	OD (260nm) / OD (280nm)	OD (260nm) / OD (230nm)
808010	30	9.33	0.75	0,3	1.87	0.06
808011	50	11,78	1	0,63	1,61	0,09
808012	17,5	10,93	0,325	0,18	0,84	0,03
808013	5	10,55	0,095	0,06	1,56	0,01
808014	22,5	12,13	0,47	0,3	1,54	0,04
1095000	37,5	18,48	0,78	0,43	1,83	0,04