Part:BBa_K4390075
C-terminal Car9-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 Car9-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). 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.
Car9 is a short silica-binding tag to add to the C-terminal of a protein using JUMP assembly, including a short glycine-rich linker (GSGGGS). 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
C-terminal Car9-tagged FAST-PETase (BBa_K4390075) was assembled by JUMP assembly with: T7 promoter (P part)-B0034 RBS (RN part) -[FAST-PETase] (O part)-[linker-Car9] (C part)-L1U1H08 (T part). All the codons were optimized for BioBrick and JUMP assembly.
Characterization
All the Lv.0 parts for [C-terminal Car9-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 Car9-tagged FAST-PETase (BBa_K4390075) PCR product (Figure 1. N1 and N2) was corresponding to 1344 bp in silico.
Figure 1. Agarose gel showed the PCR result of [C-terminal Car9-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. N: C-terminal Car9-tagged FAST-PETase. The ladder used: 1 kb DNA Ladder from NEB (N3232S).
After being transformed with the Lv.1 plasmid [C-terminal Car9-tagged FAST-PETase] into E.Coli Shuffle strain, the cells grew and were sonicated for solubility test. The weight of C-terminal Car9-tagged FAST-PETase (BBa_K4390075) should be around 33.33 kDa, which is corresponding to the red line in N2 lane (Figure 2).
Figure 2. SDS-PAGE gel showed the solubility test result of [C-terminal Car9-tagged FAST-PETase] fusion proteins. The lanes were labelled with letters, and the number behind each letter represented different fractions of the cell samples. N: C-terminal Car9-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. Shuffle: The empty Shuffle lysates without any Lv.1 plasmid. The ladder used: P7718S protein ladder from NEB
After making sure [C-terminal Car9-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 Car9-tagged FAST-PETase] showed 1.48E-03 (±1.02E-03) 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 Car9-tagged FAST-PETase showed the immobilization efficiency (100.29%), the highest over all the constructs.
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 C-terminal Car9-tagged FAST-PETase showed lower 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 209
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 438
Reference
Coyle BL, Baneyx F. A cleavable silica-binding affinity tag for rapid and inexpensive protein purification. Biotechnol Bioeng. 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.
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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