Construct plasmids with affinity tags:
Design primers for fusion tags (2 types) and signal peptides (2 types) on SnapGene, send them to a company for synthesis, determine the molecular partners to be used, and purchase the corresponding plasmids.
Amplify fragments from the vector using PCR, verify and purify the DNA fragments through agarose gel electrophoresis and gel recovery, and store them in a -20°C freezer for subsequent use.
Use Gibson assembly (one-step cloning) to connect the vector with fusion tags and signal peptide fragments.
Add the volumes of the corresponding fragments and vector, add ddH2O to make a 5 μL system, and add 5 μL of 2xClone Mix enzyme.
Heat shock transformation: Introduce the recombinant plasmids into competent cells E. coli BL21 (DE3) following specific methods and steps.
After heat shock transformation, perform preliminary screening on LB plates containing the corresponding plasmid resistance. Invert the plates and incubate at 37°C for 12 hours for colony picking and verification.
Mix the plasmids of the molecular partners with an equal amount of the original plasmid containing only LSPET.
Heat shock transformation: Introduce the plasmids into competent cells E. coli BL21 (DE3) following specific methods and steps.
After heat shock transformation, perform preliminary screening on LB plates containing the corresponding plasmid resistance. Invert the plates and incubate at 37°C for 12 hours for colony picking and verification.
Construct plasmids with mutants:
Sequencing results: Only six mutant plasmids were successfully constructed in the first attempt, so we re-constructed the remaining four mutants and finally successfully constructed ten mutant plasmids. (Refer to [experiment] for specific steps of construction)
Induce the production of the target protein, perform a series of operations such as protein extraction, disruption, and purification to obtain the enzyme solution, and react the purified enzyme with PET substrate. Liquid phase analysis showed that W159H had the best effect. In addition, IsPETaseS93_I94insE, IsPETaseT116R, and IsPETaseQ119F had higher product concentrations than the wild-type IsPETase.
Preliminary identification of transformants by colony PCR:
Pick a single colony, streak it on the corresponding resistance plate, and then place it in a prepared PCR system.
Use PCR to amplify the target fragment, verify the PCR product through DNA gel electrophoresis, select colonies with correct band sizes for sequencing verification, and save the correct strains.
Overlaying of mutant sites:
Using IsPETaseW159H as a template,overlay the other three mutation sites that enhance enzyme activity onto IsPETaseW159H.Successfully constructed IsPETaseS93_I94insE/W159H,IsPETaseT116R/W159H,and IsPETaseQ119F/W159H.
Induce the production of the target protein, perform a series of protein extraction and purification operations to obtain the enzyme solution, and react to the enzyme with PET substrate. Liquid phase analysis showed that IsPETaseQ119F/W159H had the highest enzyme activity.
Repeatedly culture and induce the bacteria with two fusion tags TrxA and NusA, two signal peptides DsbA and OmpA, and three molecular partner plasmids pTF16, pKJE7, and pGro7.
Culture → Transfer bacteria: Transfer the appropriate volume of bacterial liquid to LB medium → Induce bacteria: When the OD value is approximately 0.6-0.8, add IPTG and induce at 20°C for 19 hours.
Constructing plasmids with structural domains: (Refer to [experiment] for specific steps of the construction method)
Clone the fragments of the five structural domains from the plasmids stored in the laboratory using Polymerase Chain Reaction (PCR). Clone is from the synthesized pET22b-IsPETase plasmid. Perform double enzyme digestion on the pET-22b vector to linearize it.
Verify and recover the DNA nucleotides through agarose gel electrophoresis, including the five structural domain fragments, the linearized pET-22b vector, and IsPETase.
Fuse the five structural domains with IsPETase. Calculate the concentration of the fusion system based on the recovered gel results.
Use Gibson Assembly (one-step cloning) to connect the vector and the fused fragments. Transform the constructed five plasmids into five tubes of competent E. coli BL21 (DE3) cells, and spread the bacterial solution onto LB (AMP) solid medium for overnight incubation at 37°C.
Use T7 and reverse primers specific to the structural domains as the forward and reverse primers for colony PCR. Verify the PCR products through agarose gel electrophoresis. Mark the correct bands on the agar plate and pick single colonies from the corresponding lines to inoculate the LB liquid medium.
Send the bacterial cultures for sequencing to a company.
Using the same method as above, connect the IsPETaseQ119F/W159H mutant with the five structural domains to construct five recombinant plasmids.
Enzyme activity validation:
However, we found that after attaching the structural domains, most of the target proteins exist in a precipitated form. Therefore, we decided to introduce molecular chaperones to solve the problem of protein insolubility. Adding molecular chaperones resolved the issue of protein insolubility. The concentrations and reaction systems of the recorded enzyme solutions are shown in the figure.
Multiple rounds of protein induction were performed to produce the target protein. After a series of protein extraction and purification steps, enzyme solutions were obtained. These enzyme solutions were then reacted with different substrates. We repeated the cultivation of multiple sets of bacteria, induced multiple groups of proteins, and performed multiple rounds of reactions.
Protein extraction:
Proteins were extracted from induced bacteria expressing 2 fusion tags TrxA and NusA, 2 signal peptides DsbA and OmpA, as well as 3 chaperone plasmids pTF16, pKJE7, and pGro7.
Bacterial collection → Protein extraction: After sonication and centrifugation, the supernatant containing the cell lysate was collected, and the pellet was resuspended in urea for denaturation. We conducted multiple experiments and obtained the target proteins expressed by bacteria containing fusion tags TrxA, NusA, signal peptides DsbA, OmpA, and chaperone plasmids pTF16, pKJE7, pGro7.
Testing enzyme degradation effect:
The reaction mixture was subjected to High Performance Liquid Chromatography (HPLC) analysis to obtain the peak area of the products, analyzing the effect of different enzymes on PET degradation.