The first step to our project was designing plasmids for the purpose of surface displaying PET degrading enzymes including PETase and MHETase. To achieve our goals, we designed recombinant proteins which linked transporter proteins to the degrading enzymes. We ordered the DNA parts and designed primers through benchling. We used the DNA template and primers to perform PCR to amplify each DNA part and Gibson assembly to connect the parts into each plasmid. These plasmids were then transformed into NEB5 alpha cells for cloning or BL21 E. coli for protein expression. Successful expression was confirmed using SDS PAGE and Western Blot.

In order to test the effectiveness of the new enzymes, we used several efficient activity assays in the lab. The first was the BHET plate assay which involved using BHET, a material that would be degraded by PETase, on an agar plate to create a visual confirmation of activity. Figure 1 shows an example result for purified PETase, YeeJ-PETase, AIDA-I-PETase, and YeeJ-MHETase. The other activity assay involved placing the samples in a reaction vessel with a piece of PET for PETase or a solution of MHET for MHETase. After reacting for one full week, the samples are analyzed with HPLC to determine if the material was degraded or not.

Figure 1. BHET plate showing results for (1) purified PETase, (4) YeeJ-PETase, (7) AIDA-I-PETase, and (19) YeeJ-MHETase.

Figure 2. HPLC measurements for purified PETase and PETase constructs after 168 hours of reaction with PET film at 25 °C.

From the tests mentioned above, we found generally that surface displayed MHETase was active while PETase was less certain. In the BHET plate assay, YeeJ-MHETase gave a negative result as expected and in the HPLC measurements the peak corresponding to MHET disappeared. YeeJ-PETase and AIDA-I-PETase showed the expected positive result in the BHET plate assay but showed almost no activity based on the HPLC measurements. We theorized that the surface display efficiency of the enzymes may be too low, resulting in activities that were difficult to measure. We capitalized on the recently revealed FAST-PETase, which showed higher activity that could potentially be more easily measured with our activity assays. As in the beginning of our project, this began a new process of designing plasmids and related primers that would be used to create the newly designed constructs that were used later in the project.