Part:BBa_K4804000:Experience

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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).

User Reviews

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