Wet-Lab

First

Our wet experiments were divided into four phases. In Phase I, we completed the investigation of PET05 properties. Through two rounds of orthogonal experimental design, we determined the optimal pH, optimal NaCl concentration, optimal temperature, and suitable enzyme concentration for PET05 to exhibit PET-degrading enzyme activity.

Second

In the second stage, we synthesized and tested 40 protein sequences with potential PET-degrading enzyme functions, selected through dry experiments. The primary tasks in this stage were: first, transforming the 40 synthetic plasmids carrying the target genes into E. coli BL21 (DE3), followed by bacterial cultivation and protein purification; second, designing orthogonal experiments to test, screening, and analyzing the enzymatic activity of the 40 proteins and their optimal reaction conditions. We ultimately identified four enzymes with enzymatic activity and determined their optimal reaction conditions.

Third

In the third phase, we completed the first and second rounds of engineering cycles for PET05. We constructed, expressed, and tested 81 mutants. These 81 mutants comprised 79 single-point mutations and 2 mutations containing a disulfide bond pair, designed through two approaches. The primary tasks in this phase were: 1. Mutant construction, including primer design, PCR, homologous recombination, transformation into E. coli DH5α or BL21 (DE3), and sequencing. Second, we transformed the sequenced plasmids into E. coli BL21 (DE3), cultured the bacteria, and purified the mutant proteins. Third, we tested enzyme activity under the same reaction conditions as PET05 (55°C, 500 mM glycine, 5 M NaCl).

Fourth

In the fourth phase, we completed the third and fourth rounds of engineering cycles for PET05. We constructed, purified, and measured the activity of nearly 20 double-point combination mutants.

Our efforts yielded encouraging results. We are now proceeding with the construction and testing of double-point and multi-point combination mutants, as well as disulfide bond mutants capable of enhancing thermal stability, aiming to refine our exploration of PET05 engineering.