1. Construction of Gene Expression Vectors

We cloned the targeted genes, Fast-PETase and MHETase, into the gene expression vectors to complete its construction. The specific techniques we implemented include the enzymatic digestion of DNA plasmids, promoting DNA fragment ligation, and using Escherichia coli for transformation and cultivation.

2. Silkworm Embryo Injection and Positive Strain Screening

With the system and equipment of silkworm embryo gene injection, we introduced the successfully constructed gene expression vectors into silkworm eggs via microinjection. Based on the recombined pBac-Red and pBac-EGFP vectors, we screened positive transgenic silkworm individuals through a stereoscopic fluorescence microscope: we detected fluorescence signals in the neural tissues and eyes of silkworm embryos and adult moths. Specifically, positive transgenic silkworms were screened by observing red and green fluorescence in their eyes, indicating the presence of Fast-PETase and MHETase, respectively.

3. Detection of Recombinant Silk Protein Release

To determine whether the recombinant Fast-PETase and MHETase proteins produced in the silk secreted by the positive transgenic silkworms could be released into the buffer solution and further degrade PET plastics, we monitored the release of Fast-PETase and MHETase proteins from transgenic silk over 11 days in a KH₂PO₄-NaOH buffer solution, and then to assess their capability in degrading PET plastic.

4. Preparation of Recombinant Silk Cocoon Powder

We obtained the silk powder from the screened positive transgenic silkworms by collecting the cocoons they secreted and processing the cocoons into powder through cutting and grinding, which contain Fast-PETase and MHETase proteins. The powder containing Fast-PETase and MHETase will then be used in PET degradation tests.

5 .Evaluation of PET Plastic Degradation

We immersed an equal amount of silk cocoon powder that respectively expressed Fast-PETase and MHETase, with equivalent PET plastic samples, in a buffer solution and incubated at 50°C. This process was used to evaluate the degradation of PET plastic and to calculate the degradation rate, thus assessing the degradation efficiency of the proteins.

References

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