Proof of Concept
Welcome to iGEM 2021
In our ASTWS sewage treatment system, PETase-MHETase-Biofilm system worked together efficiently to biodegrade the PET plastic microparticles from the water. The system combined PETase, MHETase and Biofilm. PETase and MHETase are used to biodegrade microplastic particles, while Biofilm is used to capture and enrich microplastic particles from the water, so as to improve the degradation efficiency of the system.
As for whether this system can achieve the desired effect, it needs to be
proved in three aspects:
1) Whether PETase and MHETase can effectively degrade PET plastics and intermediate product MHET;
2) Whether Biofilm can capture microplastics in water and enhance the role of MHETase;
3) Whether the degradation efficiency of PETase-MHETase-Biofilm system is higher than that of the single PETase-MHETase.
For the second item, we soaked the small plastic pieces in the culture medium containing E. coli, which can express MHETase and OMP gene. After 48 hours, we observed that the biofilm produced by the bacteria (Congo red staining) was successfully attached to the surface of the plastic pieces and MHETase does not disturb the function of OMP gene (See Fig.1).
Fig.1 Biofilm produced by the engineered bacteria was successfully attached to the surface of the plastic pieces.
For the first and third items, we need to measure the degradation rates of plastics, and there are two options: to detect the quality loss of plastics, or to detect the increase of degradation products.
The result of loss of plastic mass is the most direct proof. Theoretically speaking, as long as the plastic sample of certain mass was immersed in the culture medium with engineering bacteria, it can be weighed regularly. However, in the actual experiment, we found that the biodegradation rate of plastics was relatively slow, the mass loss of plastics was quite limited in a certain period of time, and the variation of the experiment had great impact on the results. Preliminary tests have shown that minor weight reduction of the tested plastic samples can be observed, but the reliability is disputed. In order to ensure the reliability of the data, high requirements were put forward for experimental process design, experimental operation and sensitivity of weighing equipment, so as to reduce possible measurement errors and systematic errors. Thus, samples of sufficient mass should be measured regularly with a high-sensitivity analytical balance after a long enough period of time in an environment with the highest concentration of PETase-MHETase-Biofilm. Due to laboratory conditions and limited time, we abandoned this scheme.
Because of the high sensitivity of the high performance liquid chromatography (HPLC), it was more easily to detect the MHET concentration of plastic degradation products in a relatively short time by HPLC. Last year, ASTWS-China-2020 has proved that PETase can effectively degrade PET plastics to produce MHET by HPLC method. Thus we try to use the same strategy to detect the TPA, the MHET degradation product, which is catalyzed by MHETase , and then designed the following experiments.
1. Sample information
We use commercial terephthalic acid (TPA), a monomer component after MHET degradation, as standard, CAS No. : 100-21-0. The standard is provided by Sigma-Aldrich Co., LTD. PubChem No. 24850686.
Five concentration gradients were set in the experiment, which were 0/0.75/1.5/3.0/6.0 mM respectively.
There are two groups of samples to be tested.
Group No. 1 – LB Medium + E. coli containing MHETase part only + MHET (Concentration gradients: 0/0.2/0.5/1/2/4/6mM)
Group No. 2 – LB Medium + E. coli containing both MHETase and OMP part + MHET (Concentration gradients: 0/0.2/0.5/1/2/4/6mM)
Group No. 3 – LB Medium + E. coli containing PETase+MHETase part + PET (with 10 mM concentration)
Group No. 4 – LB Medium + E. coli containing PETase+MHETase and OMP part + PET (with 10 mM concentration)
1.3 Sample preparation
(1) Each group was added with commercial MHET or PET accordingly;
(2) Strains expressing MHETase were inoculated in sample No. 1;
(3) Strains expressing MHETase and OMP were inoculated in sample No. 2;
(4) Strains expressing PETase + MHETase were inoculated in sample No. 3;
(5) Strains expressing PETase + MHETase and OMP were inoculated in sample No. 4;
(6) Cultured for 30min in a shaking table at 37℃ with a rotating speed of 220rpm;
(7) Took 1ml medium from each of group and centrifuged at 15000rpm for 30 min;
(8) The supernatant of Samples No. 1 and No. 2 were taken as samples to be tested;
2. Method and experimental condition for HPLC
Instrument model: Thermo LC system
Column: Zorbax Extend C-18
Injection quantity: 20 ul
Column temperature box: 40 °C 
Flow rate: 1 mL /min 
Detection wavelength: 254nm 
Mobile phase: 70% MilliQ Water, 20% acetonitrile, and 10% formic acid
Results and Discussion
1. Standards Test
HPLC result for standard TPA concentration gradients of 0/0.75/1.5/3.0/6.0mM were shown in Fig. 2. Only the peak at 11.68 min is for TPA. The peak area size was shown in Table 1. It was very clear that the 11.68 min peak area was in direct proportion to the concentration, i.e. higher concentration of TPA caused larger peak area. (See Fig.3)
Fig.2 HPLC result of TPA Standard concentration (0.75, 1.5, 3.0, 6.0 mM)
Fig. 3 TPA peak area is in direct proportion to the concentration
2. Sample Test
No MHET and PET samples were used as controls respectively, and we could easily tell that:
1) Peak area of No MHET and PET samples were almost at same level and very low.
2) Peak areas of MHET samples were much larger than No MHET samples. Therefore, the presence of TPA in group 1 and group 2 samples indicated that the MHETase produced by E. coli in group 1 and group 2 samples has successfully degraded the MHET.
3) Peak areas of PET samples were much larger than No PET samples. Therefore, the presence of TPA in group 3 and group 4 samples indicated that the PETase-MHETase produced by E. coli in group 3 and group 4 samples has successfully degraded the PET to produce TPA.
The HPLC results of experimental samples group No.1 were shown from Fig.4.
Fig.4 HPLC result of sample group No.1 (From up to down, the MHET concentration is 0.2/0.5/1/2/4/6mM respectively)
The HPLC results of experimental samples group No.2 were shown from Fig.5.
Fig.5 HPLC result of sample group No.2 (From up to down, the MHET concentration is 0.2/0.5/1/2/4/6mM respectively)
Table 2 listed the TPA concentration calculated from TPA standard curve.
Using the same method mentioned above, we can also measure and calculate the TPA generation in sample group No. 3 (PETase + MHETase) and No. 4 (PETase + MHETase + OMP). Furthermore, based on HPLC results of sample group No. 3 and No. 4 (Table 3) OMP can not only enhance the reaction of single MHETase or PETase, but also promote the PETase-MHETase double enzyme reaction.
 Gottfried J. Palm, Lukas Reisky, Dominique Böttcher, et al. Structure of the plastic-degrading Ideonella sakaiensis MHETase bound to a substrate. Nature Communications volume 10, Article number: 1717 (2019). https://www.nature.com/articles/s41467-019-09326-3
 Furukawa, M., Kawakami, N., Tomizawa, A. et al. Efficient Degradation of Poly(ethylene terephthalate) with Thermobifida fusca Cutinase Exhibiting Improved Catalytic Activity Generated using Mutagenesis and Additive-based Approaches. Sci Rep 9, 16038 (2019). https://doi.org/10.1038/s41598-019-52379-z