First weekly team meeting:

1. Members gave brief self-introductions and were devided into five groups: wet lab, dry lab, human practice, design, and network.

2. PI and instructor gave an speech about the specifics of our laboratory.

Laboratory safety training:

1. Instructor trained us about experimental safety, including appointment of instruments, norms for the use of laboratory reagents.

2. Test on laboratory and test on experimental skills: members of wet lab need to pass the test about lab safety norms and acknowledge basic experimental skills before they are allowed access to the laboratory.

Advisor speech:

1. They introduced the basic rules of the iGEM competition, and gave us advice on project selection and project organization.

Fig. 1   Safety training

Social and documentary research:

1. We conducted research on social hotspots in the hope of discovering pressing and researchable issues around us, and we searched and read the literature on these issues.

2. We screened the project topics suggested by our members and solicited advice from PI. Finally, we determined that our the goal of project is to solve the problem of how to achieve efficient degradation of PET plastics by enzymatic degradation.

Learning laboratory operations:

1. Learn about preparation of culture medium (LB, TB, ZYM) and other reagents.

2. Learn about culturing Escherichia coli strain.

Coordination with dry lab:

1. Assist dry lab in finding and organizing protein PDB files.

Reading literature about PET degradation and PETase:

Fig. 2   Media preparation

Week 1 2024.5.6-2024.5.12

Screening of chassis cells:

We constructed recombinant plasmids and introduced them into different host cells to detect fusion protein expression.

Week 2-3 2024.5.13-2024.5.26

Constructing recombinant plasmids:

pET-21b-40-1-C-G4S-PETase,    pET-21b-40-2-C-G4S-PETase,   pET-21b-40-3-C-G4S-PETase,    pET-21b-40-4-C-G4S-PETase,   
pET-21b-50-1-C-G4S-PETase,    pET-21b-50-3-C-G4S-PETase,   pET-21b-50-4-C-G4S-PETase,    pET-21b-50-5-C-G4S-PETase.

Shaking flask-level culture:

1. We picked single colonies and incubated in LB media with 0.03 mg/mL ampicillin for 10 hours.

2. We sent samples to sequence.

Shaking flask-level fermentation:

1. We inoculated the bacterial fluid and culture for 12 hours, then vaccinated 5% bacterial suspension into 50 mL TB medium incubated for 48 hours.

2. We isolated fusion protein and determined enzyme activities.

Fig. 3   Shaking flask-level fermentation

(a). Amplification culture. (b). Precipitate of bacteria after fermentation.

Week 4-5 2024.5.27-2024.6.15

Constructing recombinant plasmids:

pET-21b-60-1-C-G4S-PETase,    pET-21b-60-2-C-G4S-PETase,   pET-21b-60-3-C-G4S-PETase,    pET-21b-60-4-C-G4S-PETase,   
pET-21b-70-1-C-G4S-PETase,    pET-21b-70-2-C-G4S-PETase,   pET-21b-70-3-C-G4S-PETase,    pET-21b-70-4-C-G4S-PETase.

Shaking flask-level culturing and sequencing

Fig. 4   The PCR identification result of pET-21b and fragment

Shaking flask-level fermentation.

Connecting pepetides to the -NH₂ terminus of PETase:

1. After pre-experiment, we found that enzyme activities were significantly different when the PET-binding peptides were connected to the -NH₂ terminus or the -COOH terminus of PETase. We tried to construct recombinant plasmids by assembling these 16 peptides at the -NH₂ and -COOH terminus of PETase.

2. Sequencing and fermentating on the shaking flask-level when sequencing correct.

Replacing linker:

1. We replaced linkers of fusion protein with SLE, AG, 10A, EAAAK, Tr. Then, we constructed recombinant plasmids.

2. Plasmids were sequencing and several were re-constructed.

Fig. 5   Construction of fusion protein

Week 6 2024.6.16-2024.6.22

Shaking flask-level fermentation:

1. Recombinant plasmids of correct sequence were fermentated for 48 hours:

pET-21b-40-1-N-SLE-PETase,    pET-21b-50-1-C-SLE-PETase,   pET-21b-50-1-N-SLE-PETase,    pET-21b-50-4-N-SLE-PETase,   
pET-21b-50-5-C-SLE-PETase,    pET-21b-70-1-C-SLE-PETase.  

2. Recombinant plasmids of wrong sequence were re-constructed and sequencing.

Enzyme activity measurement:

1. We measured enzyme activities of fusion protein by crude enzyme solution.

Fig. 6   SDS-PAGE samples

Week 7 2024.6.23-2024.6.29

Protein purification:

1. Expressible fusion proteins were purified by Ni-NTA affinity chromatography column.

2. Enzyme solution was freeze-dried after dialysis for 24 hours.

Fig. 7   Protein purification instrument

Week 8 2024.6.30-2024.7.6

PET degradation

1. Enzyme solution of G4S-liner fusion protein (protein concentration 4 μM) was added into the glass tube containing 25 mg PET amorphous particles. The degradation was reacted in 60℃ for 96 hours.

2. Samples were taken every 24 hours.

Optimization of fermentation conditions

1. We took TB, LB, and ZYM were selected as culture media to investigate the optimal medium type. IPTG concentrations of 0.025 mmol/L, 0.050 mmol/L, 0.075 mmol/L, and 0.100 mmol/L were selected to investigate the optimal IPTG concentration. 16℃ and 25℃ were fermentation temperature to investigate the optimal fermentation temperature. 24 h and 48 h were fermentation time to investigate the optimal fermentation time.

2. Then, we measured enzyme activities of fusion protein by crude enzyme solution.

Fig. 8   PET degradation samples

Week 9-10 2024.7.7-2024.7.20

EPA and MHET detection by HPLC:

1. We took PET degradation samples (24h, 48h, 72h, 96h) of week 8 to test the degradation product by HPLC.

PET degradation:

1. Expressible enzyme solution of SLE-linker fusion protein reacted with PET for 96 hours.

2. Samples were taken every 24 hours.

Week 11 2024.7.21-2024.7.27

EPA and MHET detection by HPLC:

1. We took PET degradation samples (24h, 48h, 72h, 96h) of week 10 to test the degradation product by HPLC.

2. We organized and analyzed data.

Week 12-13 2024.7.28-2024.8.10

Single spot mutation

We selected three short peptides (50-4-N-G4S, 50-5-C-G4S, 60-2-N-SLE) with better effects and performed mutant construction based on the mutation sites screened by dry lab group.

1. Mutants construction:

WildType	Mutation Sites
50-4-N-G4S	R4D, S14A, V19I, L23K, Y33F, S61A
50-5-C-G4S	I283V, R287V, C293V, R301V, A325S
60-2-N-SLE	L7F, A13S, S26D, N29D, R32K, L44I, Q55L, L60I

2. We designed primers and constructed recombinant plasmids.

Fig. 9   Construction of single spot mutants

Shaking flask-level culturing and sequencing :

1. Plasmids were sequencing and several were re-constructed.

2. Recombinant plasmids of correct sequence were fermentated for 48 hours.

Enzyme activity measurement:

1. We measured enzyme activities of fusion protein by crude enzyme solution.

Fig. 10   Fermentation samples

Week 14 2024.8.11-2024.8.17

Protein purification depending on results of enzyme activities.

PET degradation:

1. Expressible enzyme solution of mutants fusion protein reacted with PET for 96 hours.

2. Samples were taken every 24 hours.

Costruction of eGFP-fusion protein:

1. We selected pET-20b as vector, design primers to assembly eGFP and peptides (50-4-N-G4S, 50-5-C-G4S, 60-2-N-SLE) with better effects.

2. We sequenced those recombinant plasmids and re-constructed wrong plasmids.

Fig. 11   PET degradation of single spot mutants

Fig. 12   Construction of eGFP-fusion protein

(a). The PCR samples. (b). The PCR identification result of fusion protein.

Week 15 2024.8.18-2024.8.24

EPA and MHET detection by HPLC:

1. We took PET degradation samples (24h, 48h, 72h, 96h) of mutants to test the degradation product by HPLC.

Shaking flask-level fermentation:

1. Recombinant plasmids of eGFP-fusion protein were fermentated for 48 hours.

Week 16 2024.8.25-2024.8.31

Characterization of eGFP-fusion Protein

1. eGFP-fusion Protein were reacted with PET thin films at 37℃ for 24 hours.

2. Remaining fluorescence adsorption intensity of PET films are observed under a fluorescence microscope.

We organized and analyzing data.

We wrote documents and graphs.