Team:Uppsala/Experiments

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 They can therefore not be published here.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.

Laccase and dioxygenase protocols

Restriction Free Cloning

Inserting fragment A after fragment B into fragment B plasmid:

  1. Design primers for fragment A with overhangs complementary to fragment B and to the suffix of the fragment B plasmid
  2. PCR to amplify fragment A + overhangs
    • 21.2 μl ddH2O
    • 5 μl 2mM dNTP
    • 10 μl 5x HF Buffer (for Phusion polymerase)
    • 0.3 μl Phusion HF DNA Polymerase
    • 2.5 μl DMSO (total concentration 5%)
    • 10 ng plasmid with fragment A
    • 5 μl (5 ng/μl) of each primer
    • Total volume 50 μl
    • Run with PCR program below:
      • Temperature (°C) Time (hh:mm:ss) Number of Cycles Step
      95 00:02:00 1 1
      95 00:01:00 1 2
      45 00:00:45 1 3
      72 00:01:30 1 4
      GOTO step 2 5 5
      95 00:01:00 1 6
      58 00:00:45 1 7
      72 00:01:30 1 8
      GOTO step 6 29 9
      72 00:07:00 1 10
      12 Infinite time 1 11
  3. Run all of the PCR-product on a 2% agarose gel with big wells that can fit 50 μl.
  4. Extract DNA using gel extraction kit
    • 500 ng extracted product
    • 10 μl 5x HF Buffer (for Phusion polymerase)
    • 0.3 μl Phusion HF DNA Polymerase
    • 5 μl DMSO (total concentration 10%)
    • 100 ng of target plasmid (with fragment B)
    • 18.7 μl ddH2O
    • Run with the PCR program below:
      • Temperature (°C) Time (hh:mm:ss) Number of Cycles Step
      98 00:00:30 1 1
      98 00:00:20 1 2
      72 00:04:30 1 3
      GOTO step 2 15 4
      72 00:05:00 1 5
  5. Add 1 μl of Dpn1 to the PCR product and incubate for 2h.
  6. Transform with 10 μl of PCR product
  7. Colony PCR with VR and VF2, run on gel to confirm correct assembly of the parts.

Immobilized metal ion affinity chromatography (IMAC)

Materials needed
    IMAC Column; ice, stand, sample tubes.
    Sample Buffer (50mM of Sodiumphosphate, 60mM imidazol, 1 M NaCl: pH 7.1)
    Elution Buffer (50mM Sodiumphosphate, 300mM imidazol, 1M NaCl: pH7.1)
    Pump
    Ni(II)sulfate solution (20mM)
    Chelating Sepharose Fast Flow gel.

Step 1: Packing of the IMAC column.

Assemble the columns bottom part, tube and adapter. Leave the top adapter. It will be connected last to remove any bubbles.

    Degas 200ml sample buffer and roughly 200mL deionized water.
    Calibrate the pump flow rate to 1ml/min.
    Pipette carefully degassed gel into the column (the column should be filled with 1-2cm of sedimented gel).
    Fill the column carefully, without stiring the gel, with deionized water.
    Connect the last adapter and make sure there are no bubbles in the column. (Connecting it migth require some force).

Step 2: Equilibrate the column

By equilibrating the column with Ni2+ ions, the HIS-taged proteins will bind to the column.

    Run 20mL of NI(II)sulfate solution thorugh the column.
    The gel will be saturated with Ni^+2. The waste solution should be gathered in a special tube for separete disposal due to environmental reasons.
    Wash the column with roughly 20ml of deionized water. This will remove the excess of Ni+2.
    Now equilibrate the column with 30ml of degassed sample buffer.

Step 3. Running the column with sample.

    Degas 70ml of elution buffer.
    Run your sample through the column. Gather the flowthrough liquid in a marked collection tube.
    Wash the column with 30 ml of degassed sample buffer. Gather the flowthrough in 10ml fractions (Mark your fractions) . This will wash out losely bound proteins.
    Now elute your sample by running 20ml degassed elution buffert. Gather the flowthrough in 5ml fractions
    Now wash the column with 20 ml of degassed elution buffer and gather 10ml fractions. This is to make sure that all of the sample has been washed out.

Ion exchange cromatography protocol

Buffer

Prepare a phosphate buffer with a pH above or below relative to the proteins Pi.

The buffer should either positive or negativly charge the protein dependent on the charge of the Ion exchange coloumn.

Ion exchange column

Colomn Size: the size of the colomn is chosen based on the binding capacity of the gel matrix.

NaCI Gradient

Prepare a NaCI gradient to wash the columnbound protein with. Following NaCI gradient can be modified and custom made: Stock solution of NaCl with 1M diluted into 11 different 50ml falcon tubes and with one 0mM. Gradient of NaCl with concentrations:

    0 mM
    50 mM
    100 mM
    150 mM
    200 mM
    250 mM
    300 mM
    350 mM
    400 mM
    450 mM
    500 mM
    1000 mM

Step 1

Before running your cell lysis through the column it needs to be equilibrated by the chosen buffer.

    Run at least 2 column volumes 10x the concentration of the buffer.
    Run at least 2 column volumes 1x the concentration of the buffer.

Step 2

    Running sample through the column
    Run the sample through the column

Your protein is now bound to the gel matrix.

Step 3

    Wash the column with the NaCI matrix.
    Use the same amount of collection tubes as the number of different NaCl-gradients you will use.
    Run the first concentration of the NaCI gradient and collect in a marked collection tube.
    Continue running the NaCI gradient and collecting in collection tubes.

Your sample is now, hopefully and if done correctly, in one of your collection tubes.

Nah7 plasmid and Psuedomonas putida protocols

Preparation of P.putida minimal medium (PMM)

Materials required: NH4Cl, Na2HPO4·2H2O, KH2PO4 (pH 6.8), Hutner mineral base, vitamin solution

Hutner mineral base:

  • NTA
  • MgSO4·7H2O
  • CaCl2·2H2O
  • (NH4)6Mo7O24·4H2O
  • FeSO4·7H2O
  • Metals 44 solution

Metals 44 solution:

  • Na4EDTA·4H2O
  • ZnSO4·7H2O
  • FeSO4·7H2O
  • MnSO4·H2O
  • CuSO4·5H2O
  • Co(NO3)2·6H2O
  • Na2B4O7·10H2O

Vitamin solution:

  • Biotin
  • Nicitinic acid
  • Thiamin hydrochloride

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 600 µl 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 10 000 rpm 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

TLC Analysis:

  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 H2O
  • 1 ml of a trace elements solution (acidified with 37% HCl) containing 2 g FeSO4·7 H2O, 1.5 g MnSO4·H2O, 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