Difference between revisions of "Part:BBa K4390077"

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C-terminal L2NC-linker-tagged FAST-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 C-terminal L2NC-linker-tagged FAST-PETase to make the construct functional for both PET degradation and silica immobilisation.

FAST-PETase (BBa_K4390073) is an engineered mutant of PETase (290 amino acids) with (S121E/D186H/R224Q/N233K/R280A). 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). 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.

L2NC-linker is Part BBa_K3946002 but with the addition of a short linker (GSEGKSSGSGSESKST). L2NC is a truncated version of the L2 ribosomal protein from E. coli, designed for fusion to C-terminal of a protein using JUMP assembly. This tag contains just the N and C-terminal regions of L2 which were shown to have silica binding capacity in previous experiments, therefore allowing the use of a smaller tag without compromising on binding affinity. The attachment of L2NC-linker silica tag on the C-terminus of the functional enzyme would result in the 15.69 kDa increasement in weight. From literature, the dissociation constant between L2NC silica tag and silica beads is 1.7nM. Therefore, this tag facilitates immobilisation to silica surfaces, enabling enzyme immobilisation or purification using silica-based spin columns (Kim et al., 2020).

Design

C-terminal L2NC-linker-tagged FAST-PETase was assembled by JUMP assembly with: T7 promoter (P part)-B0034 RBS (R part) -[FAST-PETase] (O part) -[linker-L2NC] (C part)-L1U1H08 (T part). All the codons were optimized for BioBrick and JUMP assembly.

Characterization

All the Lv.0 parts for [C-terminal L2NC-linker-tagged FAST-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 [C-terminal L2NC-tagged FAST-PETase] PCR product (Figure 1. M1 and M2) was corresponding to 1728 bp in silico.

Figure 1. Agarose gel showed the PCR result of [C-terminal L2NC-linker-tagged FAST-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. M: C-terminal L2NC-linker-tagged FAST-PETase. The ladder used: 1 kb DNA Ladder from NEB (N3232S).

After being transformed with the Lv.1 plasmid [C-terminal L2NC-linker-tagged FAST-PETase] into E.Coli Shuffle strain, the cells grew and were sonicated for solubility test. The weight of [C-terminal L2NC-linker-tagged FAST-PETase] should be around 47.15 kDa, which is corresponding to the red line in M2 lane (Figure 2).

Figure 2. SDS-PAGE gel showed the solubility test result of [C-terminal L2NC-linker-tagged FAST-PETase] fusion proteins. The lanes were labelled with letters, and the number behind each letter represented different fractions of the cell samples. M: C-terminal L2NC-linker-tagged FAST-PETase. 1: the cell sample before sonication. 2: the supernatant of cell lysate after centrifuge. 3: the resuspended insoluble portions of cell lysate after centrifuge. TOP10: The empty E.Coli TOP10 cell lysate without any Lv.1 plasmid. The ladder used: P7718S protein ladder from NEB.

After making sure [C-terminal L2NC-linker-tagged FAST-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.

Figure 3. The protein sample activities assessment based on para-nitrophenol-butyrate pNPB assay. The reaction system was set up in 96 wells plate with final volume 10ul in each well, and the reaction continued for 30 min in 37°C (45 mM Na2HPO4-HCl (pH 7.0) 90 mM NaCl, and 10% (v/v) DMSO; 2mM pNPB-para-nitrophenol butyrate). All the constructs were constructed with T7 promoter, B0034 RBS and L1U1H08 terminator, so only the shorten forms of construct name were on the X-axis. [Shuffle without Lv.1 plasmid. 1] is the protein sample from the empty Shuffle strain of the same batch. “U” is the amount of activity which releases one micromole of pNP per minute under these assay conditions. The error bar is derived from biological triplicates.

From the first set of pNPB assay, we observed protein sample containing [C-terminal L2NC-linker-tagged FAST-PETase] showed 1.65E-03 (±2.43E-04) U activity per mg proteins towards pNPB under the reaction condition. The mean activity of biological triplicates was higher than the empty control containing no Lv.1 plasmid.

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 [C-terminal L2NC-linker-tagged FAST-PETase] showed the immobilization efficiency (11.39%), lower than the empty control’s in the same batch. One possible reason might be the positively charged L2NC would interact with negatively charged PETase surface and inhibit the immobilization activity. Meanwhile, due to the lack of LacO, the expression level of [C-terminal L2NC-linker-tagged FAST-PETase] was similar to other constitutively expressed proteins in the E.Coli Shuffle. Therefore, the low immobilization efficiency here may be due to the low level of [C-terminal L2NC-linker-tagged FAST-PETase] protein presence in the cell lysate.

After immobilization, we assessed the activity of the constructs again based on pNPB assay.

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.

Interestingly, the [C-terminal L2NC-linker-tagged FAST-PETase] showed 16.62-fold-higher activity comparing to empty control in the same batch (Figure 5). We assumed that the increasement in U/mg protein resulted from low protein loading to each silica beads. For other constructs with higher protein loading per silica bead, the enzymes may crowd together and inhibit each other’s activity. The general pattern of data indicated that the protein loading per silica bead should be well defined to maintain the enzyme activity after immobilization.

To assess the reusability of [FAST_PETase-linker-L2NC], we tested the same sample’s activity again on pNPB assay after washing out the remaining substrate and product from last reaction. Consequently, the remaining activity of [FAST_PETase-linker-L2NC] was 62.96%.

Figure 6. The comparison of [C-terminal L2NC-linker-tagged FAST-PETase] activity in first and second round of pNPB assay. The reaction system was set up in Eppendorf with final volume 1ml in each Eppendorf tube, and the reaction continued for 30 min in 37°C (45 mM Na2HPO4-HCl (pH 7.0) 90 mM NaCl, and 10% (v/v) DMSO; 2mM pNPB-para-nitrophenol butyrate). The absorbance was measured from the Spectrometer at 415nm.

We chose the immobilized [FAST_PETase-linker-L2NC] construct to continue the PET fragment degradation experiment. The heat-treated PET plastic sample was provided by Dr. Joanna Sadler. We increase the pH of reaction system since the product of terephthalic acid (TPA) would lower the pH of reaction environment. We assessed the weight loss of PET plastic fragments after reacting with immobilized PETase.

Figure 7. The comparison of PET plastic fragment before and after reaction. The fragments circled in red were the same piece of PET fragment. The fragments squared in red were the same piece of PET fragment. The number on the bottom right was the weight of the white tray. The reaction was happened in 100 mM KH2PO4-NaOH buffer (pH 8.0) in 37° C for 2 weeks.

After reaction, we took out all the PET fragments from the reaction system and dehydrated them in 37°C incubator for 2 days. We observed the fissure in the plastic fragment (red square in Figure 7), and the less irregular edges of the plastic fragment (red circled in Figure 7). The results showed that the immobilized FAST-PETase can slightly degrade the PET plastic under the reaction condition. The weight of PET fragment before reaction was 51.8 mg, and after reaction was 51.6mg. There was no significant weight loss of PET fragment, and it may result from multiple factors:

1. the optimized working temperature of FAST-PETase is 50°C, which is 13°C higher than our condition.

2. The amount of [C-terminal L2NC-linker-tagged FAST-PETase] added to the reaction was low, at most 107.34 µg in total.

3. Although the immobilized [FAST_PETase-linker-L2NC] can still react with pNPB, the C-terminus silica tag may interfere the interaction between FAST-PETase and actual PET polymer in reaction. Therefore, we supposed further assessment based on N-terminal tagged FAST-PETase should be conducted with PET sample after optimized immobilization. Considering the higher inhibition effect of N-terminal silica tag (Figure 5), we can try to substitute the N-terminal unstructured peptides (M1 to A27) with the silica tag.

4. Since the PET fragment is insoluble, PETase may react better with it in relatively static environment, instead of continuously shaking like we did.

Sequence and Features

Reference

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.