This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K3241040

In R. eutropha cells, PHB is made through 3 steps.Two acetyl-CoA molecules made from carbohydrate converted to acetoacetyl-CoA by β-ketothiolase by PhbA gene. Then acetoacetyl-CoA reductase encoded by PhbB gene catalyzes the reduction of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA (3HB-CoA). Finally, PHB synthase encoded by PhbC gene catalyzes polymerization reaction of monomer molecules to PHB.

Experiments

Determination of PHB standard curve（HPLC)

1. Accurately weigh 10 mg PHB standard into the colorimetric tube and add 5mL concentrated sulfuric acid.
2. Heat it in a 90℃ water bath for 30min.
3. Take out and cool it fully to indoor temperature.
4. Dilute the solution 100 times with 5 mmol sulfuric acid, and then use the 5mL volumetric flask to determine the volume. At this time, the content of PHB in the mother solution is 20 μL/mL.
5. Dilute the mother solution with 5mmol sulfuric acid into 0.5, 1, 2, 3, 4, 5, 6, 7, 8 ul /mL PHB solution.
6. Filter the diluted solution with a small filter（0.22μm）and take 400ul sample for measurement.
7. The filtered concentrated sulfuric acid is used as a blank control to measure.

PHB sample determination

1. Accurately weigh 10 mg dried bacteria into a colorimetric tube and add 5mL concentrated sulfuric acid.
2. Heat it in a 90℃ water bath for 30min.
3. Take out and cool it fully to indoor temperature and dilute it 100 times with 5mmol sulfuric acid.
4. Filter the diluted solution with a small filter（0.22μm）and take 400ul sample for measurement.

The conditions of HPLC

1. Mobile phase: 5mmol sulfuric acid.
2. UV detection wavelength: 210nm.
3. Flow rate: 0.6mL/min.
4. Column temperature: 60℃.

User Reviews