Part:BBa_K4390089
Untagged Tri-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 untagged Tri-PETase aiming for generating Tri-PETase with PET degradation function, and it met the requirement for the Improvement of Existing Part (BBa_K3946023)
Tri-PETase is an engineered mutant of PETase (290 amino acids) with (T140D/R224Q/N233K). 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). However, since only a very small amount of MHET can be continued to be hydrolyzed to TPA by PETase, we need to add MHETase to the device to increase TPA yield and purity in our cell-free device (Puspitasari, Tsai and Lee, 2021).
From literature search, we learnt Lu's team has enhanced the activity of PETase with CNN-based machine learning algorithms and developed FAST-PETase, the most efficient enzyme available today with five mutations comparing to wild-type PETase (S121E/D186H/ R224Q/N233K/R280A). Untreated post-consumer PET from 51 different thermoformed products is almost always completely degraded by FAST-PETase at 50 ºC for periods ranging from 24 h to 1 week. FAST-PETase can also depolymerize the untreated amorphous fraction of a commercial water bottle and an entire heat pre-treated water bottle at 50 ºC. For highly crystalline PET, a simple pre-treatment (e.g., melting) allows the PET to be feasibly degraded. We also selected another Triple mutant PETase (T140D/R224Q/N233K) with similar activity as FAST-PETase under 40°C to compare their performance (Lu et al., 2022).
Design
Untagged Tri-PETase was assembled by JUMP assembly with: T7 promoter (P part)-B0034 RBS (RN part) -[Tri-PETase] (O part) -L1U1H08 (CT part). All the codons were optimized for BioBrick and JUMP assembly.
Characterization
All the Lv.0 parts for [Untagged Tri-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 Untagged Tri-PETase PCR product (Figure 1. D) was corresponding to 1291 bp in silico.
Figure 1. Agarose gel showed the PCR result of [Untagged Tri-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. D: Untagged Tri-PETase. The ladder used: 1 kb DNA Ladder from NEB (N3232S).
After being transformed with the Lv.1 plasmid [Untagged Tri-PETase] into E.Coli Shuffle strain, the cells grew and were sonicated for solubility test. The weight of Untagged Tri-PETase should be around 31.46 kDa, which is corresponding to the red line in D 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.
After making sure [Untagged Tri-PETase] was expressing, we assessed its activity based on para-nitrophenol-butyrate (pNPB) assay, since pNPB can be hydrolysed by PETase into para-nitrophenol (pNP) with maximum absorbance at 415 nm (Pirillo, V, et al., 2021). This is a preliminary assay to determine the activity of PETase, although pNPB has structural differences to the polyethylene terephthalate which is the real substrate of PETase. Data for Tri-PETase and FAST-PETase were measured on different days (Figure 3).
Figure 3. The fold-change of 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. 3) and spectrometer. We don’t have biological replicates for constructs from [Dou_PETase] to [Tri_PETase-L2NC].
The Untagged Tri-PETase showed 1.16-fold higher activity towards pNPB over the Untagged Dou-PETase (BBa_K3946023) (Figure 3).
We immobilized the PETase mutants on the silica beads (Celite 545) after activity assessment. 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 4. 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 Untagged Tri-PETase showed the immobilization efficiency (41.02%), lower than the empty control.
Figure 5. 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 Untagged Tri-PETase showed lower activity comparing to empty control in the same batch.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.
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.
Lu H, Diaz D, Czarnecki N, Zhu C, Kim W, Shroff R et al. Machine learning-aided engineering of hydrolases for PET depolymerization. Nature. 2022;604(7907):662-667.
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