Proof of Concept
Through a p-nitrophenyl butyrate (pNPB) substrate assay and GFP visualization, we were able to demonstrate a functional proof of concept of our BioBrick device, consisting of PETase enzyme and sfGFP. This device, aside from demonstrating a proof of concept of the main objective of our project—the breaking down of PET plastic, also shows proof for a way for researchers to easily use Plastiback. To enable easy use of Plastiback, we designed our sfGFP fusion part, which can be easily visualized with ultraviolet light, thus facilitating a researcher in checking the functionality of the PETase enzyme of Plastiback. Moreover, the functionality of our device (the sfGFP fusion part and PETase) was further demonstrated with the pNPB assay discussed below.
we observed sfGFP fluorescence at a magnification of 100x. The individual specks in the following photo on the left are cells. On the right is the uninduced sample, also at 100x. Although you can still observe some baseline sfGFP fluorescence, the induced sample is much brighter and has many more cells fluorescing.
Induced (left) versus uninduced cells (right) show T7 promoter gives inducible control.
These images illustrate that our PETase-sfGFP part is being expressed. They also confirm that we have inducible control over our construct because the uninduced sample shows basal expression.
PETase fused to GFP is catalytically active on the PNPB substrate over time. Lysate induced to produce PETase was incubated with PNPB substrate (pink) or lysis buffer (green) and 405nm absorbance measured over time. The sfGFP tag produces some absorbance reading which is why the green trend line is not closer to zero. Upon the addition of PNPB substrate, absorbance increases (pink trend) suggesting that PETase is catalytically active on the PNPB substrate. Plus and minus 1 standard deviation is shown in the shaded region. Without inducer, otherwise identical cell lysate has low absorbance as it is not expressing PETase or the GFP tag (brown). Likewise, the substrate by itself has low absorbance (blue).
Our results indicate that PETase-sfGFP has background absorbance without the pNPB substrate, but that with the pNPB substrate absorbance increases, showing that PETase-sfGFP is able to catalyze pNPB into 4-nitrophenol which will absorb at this wavelength. The background is absorbance is likely due to the sfGFP fusion, which typically emits most strongly at a different wavelength but would also absorb some at 405nm. Further, this is evidence that the induction system we designed has strong control over PETase-sfGFP expression because we see almost no absorbance when the cultures were uninduced, and strong absorbance otherwise.
