Recombinant Gene Expression Vectors

First, we excised the DNA fragments of Fast-PETase and MHETase, which had been codon-optimized for Bombyx mori, from the vectors. This process is illustrated in Figure A below.

Next, we ligated these two fragments with expression vectors containing the Fibroin Heavy Chain (FHC) promoter, forming new recombinant vectors. After enzyme digestion and identification, the sizes of the ligated vectors were confirmed to be as expected. We named them pMD19T-Fast-PETase and pMD19T-MHETase, as shown in Figure B below.

Injection of Recombinant Expression Vectors into Silkworm Embryos

We first amplified the expression cassettes on the pMD19T vector-Fast-PETase and pMD19T vector-MHETase plasmids by PCR, as shown in Figure A.

Then, using homologous recombination, we recombined these two expression cassettes with the pBac-Red and pBac-EGFP vectors, respectively, obtaining clones containing the pBac-Fast-PETase plasmid and the pBac-MHETase plasmid after transformation, as shown in Figure B. This step allowed us to subsequently screen positive silkworm lines expressing plastic-degrading enzymes using a fluorescence microscope.

Next, we performed enzyme digestion identification on the obtained pBac-Fast-PETase and pBac-MHETase plasmids, and the results were as expected, as shown in Figure C.

Finally, we extracted the genomes from the selected positive transgenic silkworms and conducted PCR identification on the Fast-PETase and MHETase genes. The identification results showed the amplification of the target bands for both genes, indicating that these two genes had been successfully inserted into the silkworm genome, as shown in Figure D.

Screening of Positive Silkworm Individuals

We observed the eyes of the transgenic silkworms under a fluorescence microscope and successfully selected individuals carrying the two target genes. Based on the pBac-Fast-PETase and pBac-MHETase plasmids obtained through homologous recombination, the eyes exhibiting red fluorescence correspond to recombinant Fast-PETase silkworm individuals, while those showing green fluorescence correspond to recombinant MHETase silkworm individuals. Subsequently, we named the cocoons secreted by the silkworms carrying these two genes as RG, while the wild-type cocoons were named WT. The screening of positive silkworm individuals is shown in the figure below:

Transgenic Silkworm Silk Powder Expressing Plastic-Degrading Enzymes and Its Protein Release

We collected the cocoons secreted by the screened positive silkworm individuals and processed them into silk powder. Through scanning electron microscopy (SEM) observation, we found that the transgenic cocoons and silk powder obtained maintained the same morphological characteristics as the wild-type cocoons and silk powder, as shown in Figure A below.

In extraction experiments conducted over different time periods, we noticed changes in the release of the two enzymes over time: For Fast-PETase, its release volume was relatively high in the first 5 days, but from day 6 to day 11, its release volume decreased as the protein began to degrade. For MHETase, we observed the highest release on the first day, but the protein was completely degraded after two days. These observations are shown in Figure B below.

The observations of the enzyme release over time for both enzymes allowed us to better understand and consider which degradation scenarios the transgenic silk powder containing Fast-PETase and MHETase would be suitable for, providing more detailed insights for subsequent product development.

Testing the Degradation of PET Plastics by Transgenic Silk Powder

We selected two common PET plastic objects for enzyme degradation experiments: a non-transparent fruit container (A) and a transparent container (B), both labeled with the "PET" symbol. To facilitate the experimental process and statistical analysis, we used a hole punch to make standardized thin pieces of these two materials. The transgenic cocoons were processed into powder, which was then incubated with the PET plastic thin pieces to assess the enzymatic degradation effect. The degradation process is shown in Figure B below.

We used degradation rate and thinning rate as the two metrics to measure the effectiveness of plastic degradation. After 5 days of treatment, the transgenic silk powder achieved a 35.1% degradation rate and a 40.9% thinning rate for container A, with complete degradation achieved within 15 days. For container B, the degradation rate and thinning rate after 5 days reached 44.3% and 48.1%, respectively, with complete degradation achieved within 10 days. In contrast, wild-type silk powder did not exhibit any degradation or thinning effects on these two materials, as shown in Figure A below.

In another experiment, we incubated an equal mass of transgenic silk powder with container A material in a solution for 7 days, observing partial degradation of the material. By day 15, the material was completely degraded, and its degradation products, along with the dyes, dissolved in the water. Wild-type silk powder did not degrade the material. Similarly, after incubating transgenic silk powder with container B material for 5 days, partial degradation was observed, and by day 10, the material was completely degraded, with its degradation products dissolving in water. Wild silk powder again failed to degrade container B material.

Additionally, through scanning electron microscopy of the degradation process, we observed that during the incubation of the transgenic silk powder with these materials, both surface and internal degradation were evident. Specifically, the initially smooth and flat surface of the silk powder gradually became rough and porous, with numerous holes forming, as shown in the figure below: