Part:BBa_K3511002
Papain G23W + PueA R392F + PETase
Our team aims to construct an E. coli clone to enhance the biodegradation of plastics made from polyethylene terephthalate (PET) and polyurethane (PUR) as they are two major types of non-biodegradable plastics found in waste. Poly(ethylene terephthalate) hydrolase (PETase), a PET degrading enzyme that breaks down PET into mono-(2-hydroxyethyl)terephthalic acid (MHET) (Yoshida et al., 2016), together with two PUR- degrading enzymes, papain and polyurethanase esterase A (pueA) (Phua et al., 2004; Stern & Howard, 2000), are selected as the key enzyme components for biobrick construction. This biobrick will be introduced into E. coli to create a PET-PUR plastic-degrading superbug which will be named as “Plastilicious Coli”.
There are two main objectives in the biobrick construction: 1. To drive increased expression of plastic biodegradation enzymes in E. coli; and 2. To facilitate co-expression of three plastic-degrading enzymes to enhance PET and PUR degradation.
Regulatory components of the biobrick
To facilitate the increase in expression and stability of the three target enzymes, the biobrick design consists of a C62-T7 promoter (Part: BBa_K3511008). The C62-T7 promoter has been shown to enhance transcription of a target gene by more than 10-fold relative to the wild-type T7 promoter (Paul et al., 2012). A ribosome binding site (RBS) (Part: BBa_B0034) is inserted downstream from the promoter in the biobrick to facilitate increased protein expression.
To facilitate co-expression of the three enzymes, a recombinant GSK linker-2A peptide sequence is inserted between each enzyme gene to provide a cleavage site between the enzymes. The 2A peptide has been successfully used in an expression vector system to facilitate coexpression of multiple proteins (polyprotein system) and the dissociation of the polyprotein into discrete protein products (Amrani et al., 2004). Among the known 2A peptides, P2A (Part: BBa_K1537016) and T2A (Part: BBa_K1537017) were proven to produce the highest expression efficiency (Kim et al., 2011). The tripeptide “Gly-Ser-Gly” GSG linker is inserted in front of each 2A peptide to facilitate cleavage of the 2A peptide from the final protein products after expression (Szymczak-Workman et al., 2012).
Plastic-degrading enzyme genes in the biobrick
For PET degradation, PETase is the most well-studied PET-degrading enzyme which has been shown to have high PET degrading efficiency (Joo et al., 2018). Our team will use the PETase biobrick (BBa_K2013002) produced by the 2016 iGEM Team:UESTC-China as the PETase component for the construction of our multi-enzyme biobrick. BBa_K2013002 consists of a pelB signal peptide with a native PETase gene that can direct secretion of the protein to the bacterial periplasm and increase the stability of the gene product.
For PUR degradation, our team has further enhanced the plastic-degrading efficiency of papain and pueA by creating mutant papain G23W (BBa_K3511000) and mutant pueA R392F (BBa_K3511001) enzymes, respectively, using a rational design approach. The two mutant enzyme genes will be used together with the PETase biobrick to construct a “3-enzyme” biobrick to facilitate expression of the two PUR-degrading enzymes.
- 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 3659
- 1000COMPATIBLE WITH RFC[1000]
| None |