Team:UCL/Parts
Overview
The key enzymes in charge of fully degrading polyethylene terephthalate (PET) are PETase and MHETase [1]. PETase degrades PET and yields Mono-(2-hydroxyethyl)terephthalic acid (MHET) as product, which can be further degraded by MHETase and yield ethylene glycol (EG) and terephthalic acid (TPA). In order to ensure, efficient PET degradation in our Microbial Desalination cell (MDC), these two enzymes would need to be expressed in E. coli.
Objective
One of the goals of our Synthetic Biology group was to design a PETase-MHETase fusion protein for more efficient and coordinated degradation of PET in our MDC. For this, we had the option of expressing the protein as PETase-MHETase or MHETase-PETase. We were concerned that the order in which we expressed PETase and MHETase, could have an effect on expression, extracellular secretion and enzyme activity. Because of this, we decided to get some advice from the Mcgeehan´s group, who recently published a paper where he compared the PETase-MHETase and the MHETase-PETase fusion, with the latest one resulting more successful [2].
To measure the enzymatic activity of the fusion protein, we chose the para-Nitrophenyl Acetate (pNPA) assay [3], which determines the esterase activity of PETase as a function of increasing substrate concentration. To test the activity of MHETase, HPLC would be used to identify the presence of EG and TPA products in solution.
In order for the pNPA assay to give us meaningful results about which fusion protein has higher activity, we decided to design constructs for both positive and negative controls. The positive controls were PETase and MHETase acting as independent soluble enzymes, whereas the negative control for the assay was the empty plasmid T1_FB, which is an existing iGEM plasmid (UCL 2019).
In total, we designed 4 constructs (Fig. 1) coding for either the fusion protein or the soluble enzymes using Benchling. These were synthesised as fragments by GenScript and cloned into T1_FB using the NdeI and SpeI restriction enzymes. All 4 constructs were codon optimized for expression in E. coli K12 using the Benchling codon optimization tool, which makes use of the harmonization algorithm to account for codon usage burden as opposed to always the most frequent codon. In this way, our cells wouldn´t suffer from amino acid depletion.
Construct 1 (BBa_K3601016). PETase-MHETase fusion protein with a GS flexible linker (green) joining the C-terminus of PETase (cyan) and N-terminus of MHETase (red), and a LamB secretion peptide (pink) on the N-terminus of PETase. Construct 2 (BBa_K3601017). MHETase-PETase fusion protein with a GS flexible linker joining the C-terminus of MHETase and N-terminus of PETase, and a LamB secretion peptide on the N-terminus of MHETase. Construct 3 (BBa_K3601018). LamB secretion peptide with PETase. Construct 4 (BBa_K3601003). LamB secretion peptide with MHETase. The 4 constructs were synthesised as a fragment by GenScript and cloned into T1_FB using NdeI and SpeI restriction enzymes. They were then subsequentially transformed in E. coli cells.
A more detailed description of the 4 constructs can be found below (Fig. 2). Here we explain the role of each of the individual component.
All 4 constructs are shown in SBOL standard and have been flanked by the corresponding prefix (EcoRI and XbaI) and suffix (SpeI and PstI) of the Biobrick standard [3], in order to facilitate sharing of the constructs among the scientific community. Similarly, there are no forbidden restriction sites within the biobrick. These constructs were all codon optimized for expression in E. coli K12. The common features to all constructs were a constitutive T7 promoter, a strong RBS, the N-terminal signal peptide LamB, which ensures extracellular secretion of the enzymes via the Sec-dependent pathway, a T7 terminator and a 6x His Tag for purification. Constructs 1 and 2 are the two versions of the fusion protein that were tested with the pNPA assay to determine if changing the order of PETase and MHETase had an effect on enzyme activity. Construct 3 and 4 are the positive controls for PETase and MHETase, respectively.
- Yoshida S, Hiraga K, Takehana T, Taniguchi I, Yamaji H, Maeda Y, et al. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science (80- ). 2016 Mar 11;351(6278):1196–9.
- Knott BC, Erickson E, Allen MD, Gado JE, Graham R, Kearns FL, et al. Characterization and engineering of a two-enzyme system for plastics depolymerization. Proc Natl Acad Sci [Internet]. 2020 Sep 28 [cited 2020 Oct 26];117(41):202006753. Available from: www.pnas.org/cgi/doi/10.1073/pnas.2006753117
- Ma Y, Yao M, Li B, Ding M, He B, Chen S, et al. Enhanced Poly(ethylene terephthalate) Hydrolase Activity by Protein Engineering. Engineering. 2018 Dec 1;4(6):888–93.