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Applications of BBa_K4804000

Introduction

We have successfully constructed and tested one new composite part (PETase-R, BBa_K4804000). PETase (PET-digesting enzyme) is secreted by a bacterium (Ideonella sakaiensis 201-F6) that has the ability to degrade PET plastic (Harry P., et al., 2017). Spider silk has high strength and toughness, and has a unique three-dimensional spatial network structure, which can be used as a fixed material for some macromolecules.

Usage and Biology

We fused a spider silk protein fragment-R (a repetitive region in pyriform silk gene PySp1) to the C-terminal of PETase (Tuo Yi, 2019). p-NP (p-Nitrophenyl Butyrate) assay results showed that the PETase-R fusion protein could degrade the substrate, and HPLC analysis showed that the PET degradation efficiency was improved. In our project, we used PETase and R to build our biobrick.

Characterization

Successful protein expression

We designed the basic parts (PETase and R) by reading background literature and data, then cloning into pET-21a (+) backbone plasmid by GenScript. The composition part (BBa_K4804000) consist of PETase (BBa_K4804002) and R (BBa_K4804003) (Figure 1). The R sequence was inserted into the C-terminal of PETase using seamless cloning techniques to construct the composition part. In the end, we successfully recombined the PETase-R_pET-21a (+) plasmid, and transformed it into E.coli BL21 (DE3) strain.

The PETase-R expression was detected by SDS-PAGE after induction by IPTG. As shown in the Figure 2, compared to the simple without induction (lane 2), PETase-R induction by IPTG (lanes 3-6) have protein bands near 45 kDa, and the results indicate that the protein has been successfully expressed in BL21(DE3).

Figure 1: Constitution of T7 Promoter-RBS-PETase-R-T7 Terminator gene circuits.

Figure 2: SDS-PAGE electrophoresis analysis of PETase-R_pET-21a (+). 1: Marker; 2: Before IPTG induction simple; 3: With IPTG induction simple; 4: pellet of ultrasonic crushing with induction; 5: Flow-through solution after nickel column affinity; 6: 20mM imidazole eluent.

The fusion protein has PETase activity

The enzyme activity of PETase was performed by p-NP assay which is a common way to quantify hydrolytic activity. We selected p-Nitrophenyl Butyrate (pNPB) as the substrate, which can be hydrolyzed to p-nitrophenol (pNP) (Figure 3-A). pNP can be measured by microplate reader by the characteristic absorption at 405 nm. The bacterial supernatant after ultrasonic crushing was mixed with p-NPB, and the absorbance was measured at a series of time points. As shown in Figure 3-B, with the extension of reaction time, the OD405 value increased, which indicates that the degradation activity of the PETase-R. In addition, the p-NP assay was used to further test the effects of the degradation activity of PETase only. Figure 3-C demonstrated that, when the substrate concentration is 2 mM, the OD405 value increased with time. The OD405 value of the PETase-R system is slightly higher than PETase alone after 40 minutes. In other words, with R, PETase-R still have degradation activity.

Figure 3: Activity Test of PETase-R. (A)The mechanism of pNPB degradation; (B) OD405 of pNPB hydrolysis by overexpressed PETase-R. (C) OD405 of pNPB hydrolysis by overexpressed PETase and PETase-R.

The ability of fusion proteins to degrade PET plastics

In order to verify the degradation effect of our system on the real PET plastic, we cultured PET plastic fragments overnight in the ultrasonically-broken supernatant, and the concentration of the PET degradation product-TPA (terephthalic acid) in the solution was measured by HPLC (Figure 4-A). Figure 4-B showed the standard curve of the TPA standard. The results show that there is good linearity between TPA concentration and peak area with R^2 = 0.995. The results are shown in Figure 4-C. The relationship between peak area from HPLC results and TPA concentration was plotted to further analyze the degradation efficiency. Under the same experimental conditions, the engineered E. Coli overexpressed with PETase and PETase-R were performed to degrade the real PET microplastics. LB medium and R only solutions were set as the control groups. The results shown in Figure 5 are revealed that the peak area obtained by the PETase-R is higher than that of the PETase alone and a mixture protein (PETase and R) group.

Figure 4: HPLC results of TPA standard and real PET fragment degradation.

Figure 5: HPLC results of TPA relative concentration after proteins normalized in PET plastic degradation test.

Conclusion

The above results indicate that we have successfully constructed the prokaryotic expression system of PETase-R protein and obtained PETase-R protein by IPTG inducing. In addition, we tested the enzyme activity of PETase-R by p-NP assay, and the results showed that the C-terminal fusion of R protein did not affect the activity of PETase. The HPLC experiments on the degradation of real plastics proved that the properties of R protein might help PETase to perform the degradation function.

Reference

[1].Austin Harry P., et al. (2017) Characterization and engineering of a plastic-degrading aromatic polyesterase. Proc Natl Acad Sci USA 115: E4350–E4357.

[2].Tuo Yi (2019) Research on effects of repeat modules on properties of recombinant spidroins. Master's thesis, Donghua University.

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