Plasmid Transformation

This experiment aims to transform various target plastic degrading enzyme genes carried by the expression vector pET22b(+) into Escherichia coli BL21-Gold (DE3). This strain's highly efficient expression system will be utilized to obtain recombinant enzymes ready for subsequent activity validation. The experimental workflow lays the foundation for future protein expression and functional testing (e.g., PET degradation, colorimetric assays).

Plastic Enzyme Activity Assay

This experiment aims to transform various target plastic degrading enzyme genes carried by the expression vector pET22b(+) into Escherichia coli BL21-Gold (DE3). This strain's highly efficient expression system will be utilized to obtain recombinant enzymes ready for subsequent activity validation. The experimental workflow lays the foundation for future protein expression and functional testing (e.g., PET degradation, colorimetric assays).

SDS-PAGE and Western Blot

This experiment aims to transform various target plastic degrading enzyme genes carried by the expression vector pET22b(+) into Escherichia coli BL21-Gold (DE3). This strain's highly efficient expression system will be utilized to obtain recombinant enzymes ready for subsequent activity validation. The experimental workflow lays the foundation for future protein expression and functional testing (e.g., PET degradation, colorimetric assays).

PLA Screening Plate Preparation

This experiment outlines the protocol for preparing screening plates designed to identify strains or enzymes exhibiting Polylactic Acid (PLA) degradation capabilities. The core principle involves creating plates with an incorporated PLA emulsion, which serves as the substrate for detecting hydrolytic activity, typically visualized by clear zones. Two primary methods for plate preparation are described:

LC-MS Analysis of PLA Degradation Products

This experiment utilizes Reversed-Phase Liquid Chromatography (RP-LC) coupled with High Resolution Mass Spectrometry (HRMS) to identify low-molecular-weight products (e.g., lactic acid, oligomers) generated during PLA enzymatic hydrolysis. This analysis aims to evaluate degradation efficiency and product composition.

References

Borrowman, C. K., Bücking, M., Göckener, B., Adhikari, R., Saito, K., & Patti, A. F. (2020). LC-MS analysis of the degradation products of a sprayable, biodegradable poly(ester-urethane-urea). Polymer Degradation and Stability, 178, 109218. https://doi.org/10.1016/j.polymdegradstab.2020.109218

Hajighasemi, M., Nocek, B. P., Tchigvintsev, A., Brown, G., Flick, R., Xu, X., Cui, H., Hai, T., Joachimiak, A., Golyshin, P. N., Savchenko, A., Edwards, E. A., & Yakunin, A. F. (2016). Biochemical and structural insights into enzymatic depolymerization of polylactic acid and other polyesters by microbial carboxylesterases. Biomacromolecules, 17(6), 2027–2039. https://doi.org/10.1021/acs.biomac.6b00223

Miller, H. B., Witherow, D. S., & Carson, S. (2012). Molecular Biology Techniques: A Classroom Laboratory Manual (3rd ed.). Academic Press. https://elink.xjtlu.edu.cn/.../scope=site

Molitor, R., Bollinger, A., Kubicki, S., Loeschcke, A., Jaeger, K.-E., & Thies, S. (2020). Agar plate-based screening methods for the identification of polyester hydrolysis by Pseudomonas species. Microbial Biotechnology, 13(1), 274–284. https://doi.org/10.1111/1751-7915.13418

Teeraphatpornchai, T., Nakajima-Kambe, T., Shigeno-Akutsu, Y., Nakayama, M., Nomura, N., Nakahara, T., & Uchiyama, H. (2003). Isolation and characterization of a bacterium that degrades various polyester-based biodegradable plastics. Biotechnology Letters, 25(1), 23–28. https://doi.org/10.1023/A:1021713711160