Part:BBa_K4390087

N-terminal Car9-tagged MHETase

This part is not compatible with BioBrick RFC10 assembly but is compatible with the iGEM Type IIS Part standard which is also accepted by iGEM.

Usage and Biology

We designed the N-terminal Car9-tagged MHETase to make the construct functional for both MHET hydrolysis and silica immobilisation.

MHETase is an enzyme discovered in Ideonella sakaiensis at the same time as PETase (Yoshida, 2016). MHETase can hydrolyse Mono-(2-hydroxyethyl) terephthalic acid (MHET), the major product of PETase, further to terephthalic acid (TPA) and ethylene glycerol (EG). An advantage of using MHETase is that PETase degrades PET into MHET majorly, and MHET had a higher inhibitory effect on the overall hydrolysis of PET since it also originated from the other reaction product, that is, from BHET. Since TPA and EG did not affect the enzyme hydrolysis of PET, the observed inhibition caused by MHET and BHET is probably due to their ester bonds that occupy the TfCut2 substrate binding site (Pirillo, V, et al., 2021). From literature, there is a ∼60 Å long intrinsically disordered tether structure (residues 1–25) at the N-terminus of the MHETase (Pinto et al., 2021). Car9 is a short silica-binding tag to add to the N-terminal of a protein using JUMP assembly, including a short glycine-rich linker (AAAL). The tag facilitates immobilisation to silica surfaces with a dissociation constant (1 µM), enabling enzyme immobilisation or purification using silica-based spin columns. The advantage of using Car9 silica tag is its small size (1.87 kDa) would introduce smaller effect to the functional enzyme activity in theory.

Design

N-terminal Car9-tagged MHETase was assembled by JUMP assembly with: T7 promoter (P part)-B0034 RBS (R part)-[Car9-linker] (N part)-MHETase (O part)-L1U1H08 (CT part). All the codon is optimized for BioBrick and JUMP assembly.

Characterization

All the Lv.0 parts for [N-terminal Car9-tagged MHETase] were integrated into pJUMP29-1A(Laz), which is a JUMP Lv.1 backbone plasmid. The Blue-White screening was conducted to select the correct colony. The colony PCR was used to verify the band size of colony PCR product was the same as in silico simulation. The primers used were (PS1: AGGGCGGCGGATTTGTCC; PS2: GCGGCAACCGAGCGTTC), the general primers for all JUMP plasmids to amplify the insertion DNA. The size of N-terminal Car9-tagged MHETase PCR product (Figure 1. V1 and V2) was corresponding to 2280 bp in silico.

Figure 1. Agarose gel shows the PCR result of [N-terminal Car9-tagged MHETase] (agarose concentration 1.2%). The lanes are labelled with letters, and the number behind each letter represent different colonies from Blue-White Screening. V: N-terminal Car9-tagged MHETase. S: Untagged MHETase. The ladder used: 1 kb DNA Ladder from NEB (N3232S).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Reference

Yoshida S, Hiraga K, Takehana T, Taniguchi I, Yamaji H, Maeda Y et al. A bacterium that degrades and assimilates poly (ethylene terephthalate). Science. 2016;351(6278):1196-1199.

Pirillo V, Pollegioni L, Molla G. Analytical methods for the investigation of enzyme‐catalyzed degradation of polyethylene terephthalate. The FEBS Journal. 2021;288(16):4730-4745.

Pinto A, Ferreira P, Neves R, Fernandes P, Ramos M, Magalhães A. Reaction Mechanism of MHETase, a PET Degrading Enzyme. ACS Catalysis. 2021;11(16):10416-10428.