To determine whether any of the silkworm individuals microinjected with the transgenic expression vectors are successfully expressing the Fast-PETase and MHETase proteins, which will allow for a qualitative assessment of the results of our early-stage experiment as well as a screening for the positive transgenic silkworm individuals, the gene fragments for Fast-PETase and MHETase were subcloned into the p[3xp3 DsRed SV40] and p[3xp3 EGFP SV40] transgenic expression vectors, respectively. These vectors contain the 3xp3-DsRed-SV40 and 3xp3 EGFP-SV40 transcriptional regulatory units, which enable the specific expression of red and green fluorescent proteins in the eyes and nervous system of the silkworms, emitting visible red and green fluorescent signals. We can then screen positive individuals by observing red or green fluorescence in their eyes, thus allowing us to identify and establish the transgenic silkworm strain.

The silk secreted by silkworms that express both recombinant Fast-PETase and MHETase proteins was as “RG silk”. We analyzed and measured the release of recombinant Fast-PETase and MHETase proteins in RG silk by continuously extracting the recombinant proteins via the KH2PO4-NaOH buffer. The specific methods are as below:

1. Weighing 30 mg of RG silk sample; mixing the sample with 1 mL of KH2PO4-NaOH buffer (100 mM KH2PO4-NaOH, pH = 8.0). The mixture was incubated at 37°C for 1-11 days.

2. Collecting the protein samples by centrifuging the mixture at 13,400 rpm for 5 minutes at 4°C. The release of recombinant Fast-PETase and MHETase proteins in the RG silk extract can be measured by SDS-PAGE and Western blotting.

3. The protein samples were subjected to 12% SDS-PAGE electrophoresis and stained with Coomassie Brilliant Blue R-250.

4. In the Western blotting experiment, the protein samples from the SDS-PAGE gel were transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% fat-free milk buffer for 1 hour, incubated with Flag primary antibody buffer (Beyotime, China) for 1 hour, washed 5 times with TBST buffer, incubated with IgG secondary antibody buffer (Beyotime, China) for 1 hour, and washed 5 times with TBST buffer.

5. Photographing and recording the results by a Chemiscope Series instrument (Clinx Science Instruments, China) with automatic exposure.

In this experiment, we selected two types of PET plastic products for the experimental plastic sample: the opaque household plastic product A, and the transparent plastic product B.

1. Using a hole punch to produce plastic sheets from products A and B into with a diameter of 4.5 mm as the experimental samples.

2. Weighing the samples A and B by an analytical balance with their initial mass recorded as M0.

3. Measuring the samples A and B' thickness with a vernier caliper, with the initial thickness recorded as H0.

4. Immersing the samples A and B in KH2PO4-NaOH buffer containing equal amounts of RG silk powder (30 mg/mL). Treating the mixture continuously at 50°C for 15 days.

5. The degradation of PET plastics by RG silk powder can be evaluated by analyzing the degradation rate, thinning rate, and surface morphological changes of plastic samples A and B.

Degradation rate (%) = ((M0 - Mx) / M0) * 100

M0: Initial weight of the plastic product; Mx: Weight of the plastic product after X days of degradation.

Thinning rate (%) = ((H0 - Hx) / H0) * 100

H0: Initial thickness of the plastic product; Hx: Thickness of the plastic product after X days of degradation.