Difference between revisions of "Part:BBa K4390090"

(Usage and Biology)
(Reference)
 
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==Reference==
 
==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.
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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).
 
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.
 
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.

Latest revision as of 14:02, 12 October 2022


Untagged 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 untagged FAST-PETase aiming for generating FAST-PETase with PET degradation function, and it met the requirement for the Improvement of Existing Part (BBa_K3946023)

FAST-PETase (BBa_K4390073) is an engineered mutant of PETase (290 amino acids) with (S121E/D186H/R224Q/N233K/R280A). 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). 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 (Lu et al., 2022). For highly crystalline PET, a simple pre-treatment (e.g., melting) allows the PET to be feasibly degraded.

Design

Untagged FAST-PETase was assembled by JUMP assembly with: T7 promoter (P part)-B0034 RBS (RN part) -[FAST-PETase] (O part)-L1U1H08 (CT part). All the codons were optimized for BioBrick and JUMP assembly.

Characterization

All the Lv.0 parts for [Untagged 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 Untagged FAST-PETase PCR product (Figure 1. J) was corresponding to 1291 bp in silico.

PCR FAST6.png

Figure 1. Agarose gel showed the PCR result of [Untagged 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. J: Untagged FAST-PETase. The ladder used: 1 kb DNA Ladder from NEB (N3232S).


To make sure [Untagged 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.

TABLE1 FAST6.png

Figure 2. 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.


The Untagged FAST-PETase showed 1.96-fold higher activity towards pNPB over the Untagged Dou-PETase (BBa_K3946023) (Figure 2).

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.

TABLE2 FAST6.png

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 Untagged FAST-PETase showed the immobilization efficiency (34.47%), higher than the empty control.

TABLE3 FAST6.png

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 Untagged FAST-PETase showed lower activity comparing to empty control in the same batch.


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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 209
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 438

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).

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.