Difference between revisions of "Part:BBa K4390085"

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N-terminal Car9-tagged Dou-PETase

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 Dou-PETase to make the construct functional for both PET degradation and silica immobilisation.

Dou-PETase (BBa_K3946023) is an engineered mutant of PETase (290 amino acids) with (W159H/ S238F) (Austin et al, 2018). PETase was discovered in 2016 in Ideonella sakaiensis, which uses PET as a single carbon source (Yoshida, 2016). The PETase hydrolyses PET polymers and produces mono(2-hydroxyethyl)-TPA (MHET) majorly, and minorly two final products shown below: terephthalic acid (TPA), and ethylene glycol (EG) (Joo et al., 2018).

Car9 is a short silica-binding tag to add to the N-terminal of a protein using JUMP assembly, including a short alanine-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 (Coyle and Baneyx, 2014).

Design

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

Characterization

All the Lv.0 parts for [N-terminal Car9-tagged Dou-PETase] 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 Dou-PETase PCR product (Figure 1. C) was corresponding to 1338 bp in silico.

Figure 1. Agarose gel showed the PCR result of [N-terminal Car9-tagged Dou-PETase] fusion proteins (agarose concentration 1.2%). The lanes were labelled with letters, and the number behind each letter represented different colonies from Blue-White Screening. C: N-terminal Car9-tagged Dou-PETase. The ladder used: 1 kb DNA Ladder from NEB (N3232S).

After being transformed with the Lv.1 plasmid [N-terminal Car9-tagged Dou-PETase] into E.Coli Shuffle strain, the cells grew and were sonicated for solubility test. The weight of N-terminal Car9-tagged Dou-PETase should be around 33.33 kDa, which is corresponding to the red line in C lane (Figure 2).

Figure 2. The solubility test result of different constructs. The soluble portions of each construct cell lysates after centrifuge were load on the gel. The lanes were labelled with letters representing different constructs. C: T7Pro-B0034-Car9-[Dou-PETase]-L1U1H08. D: T7Pro-B0034-[Tri-PETase]-L1U1H08. E: T7Pro-B0034-[L2NC-linker]-[Tri-PETase]-L1U1H08. F: T7Pro-B0034-Car9-[Tri-PETase]-L1U1H08. G: T7Pro-B0034-[Tri-PETase]-[L2NC-linker]-L1U1H08. H: T7Pro-B0034-[Tri-PETase]-Car9-L1U1H08. I: T7Pro-B0034-[Tri-PETase]-L2NC-L1U1H08. The ladder used: P7718S protein ladder from NEB, and the range of constructs weight was labelled.

We immobilized the [N-terminal Car9-tagged Dou-PETase] on the silica beads (Celite 545). The immobilization was done by incubating cell lysate with silica beads on a rotating shaker for 30 mins, 4°C. The protein concentration was measured by Bradford assay before and after incubation.

Figure 3. The immobilization efficiency of different PETase constructs after 30mins incubation in 4°C. Immobilization efficiency= ([initial protein] - [protein in the washing buffer]) / [initial protein]. The protein concentration in the beginning solution and in the washing buffer was measured by Bradford assay. We load 500ug protein sample to each 20mg Celite545 silica beads for all constructs. [Shuffle without Lv.1 plasmid.2] was the protein sample from the empty SHuffle strain of the same batch for Tri-PETase. [Shuffle without Lv.1 plasmid.3] was the protein sample from the empty Shuffle strain of the same batch for FAST-PETase.

The N-terminal Car9-tagged Dou-PETase showed the immobilization efficiency (46.65%), higher over the empty control.

Figure 4. The fold-change of immobilized protein samples activity over the activity in empty control from the same batch. The fold changes of activity from [Dou_PETase] to [Tri_PETase-L2NC] were calculated by [activity of experimental group]/[SHuffle without Lv.1 plasmid.2]. The fold changes of activity from [FAST_PETase] to [FAST_PETase-L2NC] were calculated by [activity of experimental group]/[SHuffle without Lv.1 plasmid.3] The error bars on column from [FAST_PETase] to [FAST_PETase-L2NC] were calculated by data from plate reader (Figure. 2) and spectrometer (Figure.4). We don’t have biological replicates for constructs. [Shuffle without Lv.1 plasmid.2] was the protein sample from the empty SHuffle strain of the same batch for Tri-PETase. [Shuffle without Lv.1 plasmid.3] was the protein sample from the empty Shuffle strain of the same batch for FAST-PETase.

The N-terminal Car9-tagged Dou-PETase showed no activity comparing to empty control in the same batch. We assumed that the protein loading on each silica bead should be low. If not, the enzymes may crowd together and inhibit each other’s activity. The data indicated that the protein loading per silica bead should be well defined to maintain the enzyme activity after immobilization, especially for Car9 silica tag which showed very strong binding activity to silica beads.

Sequence and Features

Reference

Austin H, Allen M, Donohoe B, Rorrer N, Kearns F, Silveira R et al. Characterization and engineering of a plastic-degrading aromatic polyesterase. Proceedings of the National Academy of Sciences. 2018;115(19).

Coyle BL, Baneyx F. A cleavable silica-binding affinity tag for rapid and inexpensive protein purification. Biotechnol Bioeng [Internet]. 2014 Oct 1;111(10):2019–26.

Joo S, Cho I, Seo H, Son H, Sagong H, Shin T et al. Structural insight into molecular mechanism of poly(ethylene terephthalate) degradation. Nature Communications. 2018;9(1).

Puspitasari N, Tsai S, Lee C. Class I hydrophobins pretreatment stimulates PETase for monomers recycling of waste PETs. International Journal of Biological Macromolecules. 2021;176:157-164.