Results

In this section, we will present all the key results that have been critical in advancing our project. These results include plasmid and mutant construction, enzyme efficiency assay in E.Coli and cyanobacteria, signal peptide construction, PETase expression, transformation into Syn. PCC 7942 and co-culture of Rhodococcus jostii and cyanobacteria.

We identified several PETases from the iGEM distribution kit, which are the first naturally evolved PETase from Ideonella sakaiensis and their mutants. To estimate PETase enzyme activity, we followed 2019 Toronto iGEM team’s method of measuring esterase degradation using p-Nitrophenol butyrate (from Part:BBa K2910000 - parts.igem.org). The enzymatic activity of these IsPETases were measured and estimated accordingly.

The relative enzyme efficiency (A415/protein concentration) that we are looking at takes into consideration both the efficiency of the enzyme itself and the PETase synthesis rate of the chassis, since our end goal is to implement the engineered organism in a self-sufficient PET degrading system as a whole.

After reviewing several papers predicting PETase protein efficiency and activity, we identified mutation sites in IsPETase that could potentially enhance activity. We also found a PETase with higher predicted activity than wild type IsPETase — BhrPETase. We utilized an AI-assisted protein design tool based on methods described in literature. Using the vector pET28a, we constructed plasmids containing our PETase sequences. The sequence is inserted between the T7 promoter and T7 terminator.

We tested whether our plasmid constructions were successful using gel electrophoresis and sequencing.

Enzyme Efficiency assay in E.coli

Figure 6. The graph depicts the enzymatic activity of all the mutation of IsPETase,(including the ones we generated using AI and those found in the iGEM distribution kit) compared to that of WT IsPETase. The results demonstrate that our mutated variant S12E/R224Q/N233K has a higher enzymatic activity than the highest found in the iGEM distribution kit (BBa_K2910000).

Figure 7. The image shows the enzyme activities of BhrPETase WT and IsPETase WT compared to the mutations N191S, M57L, W229F, N205G. The mutation N205G has a slight higher enzyme activity than BhrPETase WT at 30 min, and has certain potential to significance surpass the efficiency of BhrPETase if given more time, but the enzymatic activities of W229F, M57L, N191S are less than IsPETase WT which indicates they have a lower efficiency.

We used ultrasonic to lyse the E. coli cells to release the PETase so that they can decompose plastic found outside of the cell. However, not only does the chassis die, the enzymes also lose their bioactivity when released this way. Hence, signal peptides are needed to allow the enzyme exit the double E. coli’s double membrane without disturbing the cell. We inserted the signal peptide pelB sequence before the enzyme sequence. The newly constructed plasmids were verified through sequencing.

PETase expression assay in E.coli

To verify IsPETase S12E/R224Q/N233K and BhrPETase N205G expression in E.coli, we performed SDS-PAGE to check the protein expression of wild type IsPETase (~30 kDa) after inducing with IPTG. (see protocol)

Figure 9. the image shows the results from SDS-PAGE. Cell Culture: represents the protein obtained from cells. Cell Culture Medium: represents the proteins obtained from the supernatent after centrifuge.

The results demonstrate the successful expression of our enzymes in E.coli and its secretion into the extracellular matrix.

Scanning Electron Microscope

We used a Scanning electron microscope to observe whether the plastic was degraded by PETase (IsPETase S12E/R224Q/N233K and BhrPETase N205G).

Figure 10. SEM photograph of surface changes of PET microplastics. The results show that the engineered bacteria can degrade the neck of plastic bottles with low crystallinity, but cannot degrade plastic films with a possible high level of crystallinity.
(a) Neck of plastic bottles (crystallinity≈1.2%) treated with E.coli harbored with BhrPETase-N205G for 12 days, control is treated with E.coli pET28a,
(b) neck of plastic bottles(crystallinity≈1.2%) treated with E.coli harbored with IsPETase-S121E/R224Q/N233K for 12 days, control is treated with E.coli pET28a,
(c)Plastic film (crystallinity unknown) treated with IsPETase.

Natural transformation of Synechococcus elongatus PCC 7942

For expression of IsPETase S12E/R224Q/N233K and BhrPETase N205G in cyanobacteria, we reconstructed plasmids using a transfer vector. The plasmids were transformed into Synechococcus elongatus PCC 7942. Successful transformations were selected using the antibiotic spectinomycin.

Figure 11. Petri dishes containing transformed cyanobacteria

Figure 12. The two petri dishes are transformed IsPETase –S121E/R224Q/N233K and BhrPETase-N205G. 3 single colonies were identified for each.

Enzyme efficiency assay

We applied the p-Nitrophenyl Butryte degradation assay to test enzyme efficiency of those expressed in cyanobacteria. (see protocol)

Figure 13. This graph depicts the enzyme efficiency of those expressed in cyanobacteria. Although the overall enzyme activities are low, BhrPETase-N205G’s activity is relatively higher than the other sample.

We think these results are possibly due to (1) signal peptides do not perform well in cyanobacteria and (2) cyanobacteria has a relatively slower growth than E.coli; therefore, we need to grant it more time to grow.

Analysis of Degradation Product

HPLC is a method to directly detect the presence of specific compound in a chemical mixture. During the degradation of PET by PETase, TPA is created as a byproduct; hence, the presence of TPA in the final product indicates degradation occurred. (see protocol)

Figure 14. The results show that TPA is present after a 2 week incubation of PET with the engineered bacteria.

Rhodococcus jostii and cyanobacteriaco-culture

Growth Medium Optimization

Cyanobacteria has a slower growth rate compared to that of Rhodococcus, which is relatively fast. Therefore, the ratio of the cyanobacteria growth medium, BG11, and that of Rhodococcus, R2A, need to be optimized in order to achieve a balanced growth of both organisms. Growth of the organisms was observed in growth mediums with a 1:3, 1:2, and 1:1 BG11 to R2A ratio. Through observing growth in both liquid and solid media, we determine that the cyanobacteria and rhodobacteria co-culture achieves optimal growth in a 1: 3 ratio growth medium.

Figure 15. The image shows the solutions of cyanobacteria and medium which different ratio, 1: 3, 1:2, 1:1.

Figure 16. The image shows the growth of cyanobacteria and Rhodococcus on cultural medium plate, ratio 1: 3.