Difference between revisions of "Part:BBa K4390088"

Latest revision as of 13:58, 12 October 2022

Untagged Dou-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

Dou-PETase (BBa_K3946023) is an engineered mutant of PETase (290 amino acids) with (W159H/ S238F) (Austin et al, 2018). 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).

We designed the untagged Dou-PETase aiming for produce Dou-PETase with both PET degradation function, and it’s also a control for the Improvement of Existing Part (BBa_K3946023)

Design

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

Characterization

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

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

To make sure [Untagged Dou-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). Active PETase can produce a yellow colour visible to the naked eye, and the absorbance can be measured on spectrometer on 415nm. 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, and so-called Batch #2 and Batch #3, respectively (Figure 2).

Figure 2. 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 and spectrometer. We don’t have biological replicates for constructs from [Dou_PETase] to [Tri_PETase-L2NC].

The Untagged Dou-PETase showed 86% activity of Untagged Tri-PETase and 55% activity of Untagged FAST-PETase towards the pNPB under the reaction condition (Figure 2).

Sequence and Features

Reference

Austin H, Allen M, Donohoe B, Rorrer N, Kearns F, Silveira R et al. Characterization and engineering of a plastic-degrading aromatic polyesterase. Proceedings of the National Academy of Sciences. 2018;115(19).

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).