Team:Imperial College/3HB assay

From 2013.igem.org

3HB assay #1

This experiment completes the cycle of PHB bioplastic recycling.

Please see 3HB protocol for sample preparation and the way we adapted the kit for our purposes.

The well setup was as shown below:

We prepared serveral dilutions of the samples and measured the absorbance at 445, 450 and 455.

Collected_data_for_3HB_assay.JPG

We corrected the absorbance values busubstracting the 0 standard.

Data_collection.JPG

We plotted the standard curve and used the below formula to calculate the 3HB concentrations in our samples.

Formula_for_calculation.JPG

3HB assay data new.JPG

We have taken the dynamic range of the assay into account (according to protocol) and therefore excluded readings where the concentration of 3HB in the well was above 0.5 mM because these are not accurate.

Imperial_P3HB_from_waste.jpg


3HB assay #2

I have added 20uL of phaZ1 purified enzyme solution (0.36 ug/uL) to the material purified from various waste-cultures, in 150 uL total volume. I used 100mM phosphate buffer only for the samples without phaZ1 enzyme. I left all the samples in the shaking incubator at 37°C for 5 hours. I measured the 3HB concentration in triplicates on the well-plate, in 13% and 1.3% dilution.

Full annotated diagram.jpg

I have measured again the hybrid-PHB without and with phaZ1 and discovered that there was a 4-fold increase in the amount of 3HB released after phaZ1 treatment.

3HB assay #3

3HB_assay_permease.JPG

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