Experiments and protocols

A majority of the protocols we used originate from the book "Synthetic biology-A lab manual", written by Anthony Forster, Erik Gullberg and Josefine Liljeruhm. They can therefore not be published here. We do however recommend you to buy it, as it was a great help in our project. The following protocols from the sixth chapter of this book were used:

1. 0.9% NaCl
2. 50% Glycerol
3. 1M CaCl₂
4. 10x TBE Buffer
5. SOB Medium
6. LB Medium
7. LB Agar Plates and Addition of Antibiotics
8. Overnight Cultures with antibiotics, and glycerol stocks
9. Agarose gel electrophoresis
10. Preparation of competent E. coli cells using CaCl₂
11. Transformation of CaCl₂ competent E. coli cells
12. Bacterial re-streak techniques
13. Digestion with DpnI
14. 3A assembly
15. Colony PCR

We also used several other protocols which we share below.

Biosurfactant characterization protocols

Drop collapse test

Materials required: 50mm petri plates, stop watch, olive oil and bacterial culture

Method:

1. A 50mm petri plate was covered with 600µl of olive oil.
2. In the centre of the plate, small drops of either 50 or 100µl of bacterial culture were placed.
3. The drop was observed for eventual collapse and the diameter of the drop was measured after 0, 5, 10, 15 and 20 min.
4. A collapsed drop indicate that the presence of biosurfactants.

Thin Layer Chromatography

Materials required: Bacterial culture, 0.45 syringe filter, ethyl acetate, ethanol, chloroform, methanol, acetic acid, orcinol, sulphuric acid, hot air oven , vacuum centrifuge and TLC silica plates

Sample preparation:

1. Overnight bacterial culture was centrifuged 10000rpm for 10 minutes.
2. Supernatant of the samples were filtered using 0,45 μm syringe filter.
3. The filtered supernatant was extracted with ethyl acetate in 1:1 v/v ratio three times.
4. The organic solvent was removed by evaporation using vacuum centrifuge overnight.
5. 10 μl 99% ethanol was added to the dried samples which then could be loaded on TLC silica plates

Sample preparation:

1. TLC was performed using chloroform/methanol/acetic acid in a ratio of 65:15:2 as a developing solvent.
2. For visualisation, the plate that has been developed was air dried and sprayed with a detection agent composed of 0.075 g orcinol, 4.1 mL sulphuric acid (60 %, v/v) and 21 mL deionised H₂O.
3. The plate was left to dry at room temperature, and then, the sugar moieties were stained by incubating the plates at 110 °C for 10 min.

CTAB plate recipe

Agar plate mineral salts medium: for 500 ml (pH 6.7)

• 10 g carbon source peptone per liter of distilled water
• 0.35 g KH₂PO₄
• 0.45 g Na₂HPO₄
• 1 g NaNO₃
• 0.2 g MgSO₄*7 H₂O
• 0.05 g CaCl₂*2 H₂0
• 1 ml of a trace elements solution (acidified with 37% HCl) containing 2 g FeSO₄*7 H₂O, 1.5 g MnSO₄*H₂0, and 0.6 g (NH₄)₆Mo₇O₂₄*4H₂O per-liter distilled water.

CTAB-methylene blue agar:

• 0.1g CTAB (cetyl-trimethyl-ammonium bromide)
• 0.0025 g methylene blue
• 7.5 g agar (difco) to 500 ml salts medium

Method: Preparation is similar to the preparation of LB agar plates