Part:BBa_K3946023
PETase (W159H/ S238F)
Double mutant PET hydrolase capable of degrading PET polymer.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 625
- 1000COMPATIBLE WITH RFC[1000]
Improvement by Edinburgh-UHAS_Ghana 2022
We improved upon the Dou-PETase part (Part:BBa_K3946023) with the FAST-PETase part (Part:BBa_K4390073).
PETase-silica tag fusion protein Activity Test
We assessed the various PETase activities using a para-nitrophenol-butyrate (pNPB) assay, since PETases can hydrolyse pNPB into para-nitrophenol (pNP), which strongly absorbs at 415 nm. pNPB is not the PETase’s true substrate, but this preliminary assay is still representative of PETase activity. Figure 1 shows that untagged FAST-PETase has higher activity than Dou-PETase in this assay. Hence, FAST-PETase is an improvement over Dou-PETase for degradation of PET. The data also shows that PETase activity is diminished, but still exists when immobilised on silica beads, as the activity is higher than SHuffle E. coli lysate.
Contribution from Edinburgh-UHAS_Ghana 2022
Plasmid assembly
All the Lv.0 parts for PETase (W159H/ S238F), which we called Dou-PETase in the following text, 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 Dou-PETase (BBa_K3946023) PCR products (Figure 1. A1 and A2) was corresponding to the in silico band size, 1291 bp.
Figure 1. Agarose gel showed the PCR result of Dou-PETase (agarose concentration 1.2%). The lanes were labelled with letters, and the number behind each letter represented different colonies from Blue-White Screening. A: Dou-PETase. The ladder used: 1 kb DNA Ladder from NEB (N3232S).
Activity assessment
To make sure Dou-PETase was expressed, soluble and active, 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. To make systematic comparison between Dou-PETase and other PETase related construct in our project, we calculate the fold-change of protein sample activity towards pNPB over the empty control from the same batch.
The Dou-PETase showed 2.35-fold higher activity towards pNPB under reaction conditions. And it shows 86% activity of Untagged Tri-PETase and 55% activity of Untagged FAST-PETase towards the pNPB under the reaction condition (Figure 2).
Figure 2. The protein sample activities result based on para-nitrophenol-butyrate pNPB assay. The figure presented 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 reaction system was set up 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. [Shuffle without Lv.1 plasmid.2] was the protein sample from the same batch of the empty SHuffle strain as Tri-PETase constructs. [Shuffle without Lv.1 plasmid.3] was the protein sample from the same batch of the empty SHuffle strain as FAST-PETase constructs. “U”is the amount of activity which releases one micromole of pNP per minute under these assay conditions. The activity of [Shuffle without Lv.1 plasmid.2] is 1.24E-03 U/mg protein sample. The activity of [Shuffle without Lv.1 plasmid. 3] is 4.44E-03 U/mg protein sample.
References
Pirillo V, Pollegioni L, Molla G. Analytical methods for the investigation of enzyme‐catalyzed degradation of polyethylene terephthalate. The FEBS Journal. 2021;288(16):4730-4745.
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