Results
The enzyme PETase plays a crucial role in degrading PET micro particles. With microplastics being highly populated in marine environments, we decided to synthesize the PETase gene into plants. Plants naturally absorb water up into their systems to photosynthesize, and are therefore a suitable host for us to synthesize the enzyme PETase into. The bioengineering of significant genes to combat global issues has become a hot area of research, especially for mediating serious issues like microplastics. This research aims to engineer PETase into Nicotiana benthamiana as a proof of concept for synthesizing PETase into plants.
Beginning the process, N Benthamiana was transformed using the PGWB2 and pCAMBIA1302 vectors, which contained the key genes responsible for the expression of the PETase enzyme and the localization of the enzyme -- PETase gene and the αAmy3SP signal peptide. These genes are then introduced into N. benthamiana to show that PETase can successfully be transformed into a plant alongside with being localised in the extracellular space, as microplastic particles are too large to enter the cell.
Transformation into Agrobacterium
After transformation, PCR amplification and gel electrophoresis were performed to confirm the successful insertion of the PETase gene in the PGWB2 and pCAMBIA1302 vectors. The presence of expected bands verified the genetic transformation in our engineered construct, and is also a key step before moving forward to the next step.
Fig.1 Gel electrophoresis following PCR amplification for PGWB2 vector, showing the detection of the IsPETase-Amy3S gene we inserted.
Fig.2 Gel electrophoresis following PCR amplification for pCAMBIA1302 vector, showing the detection of the IsPETase-Amy3S gene we inserted.
As shown in Figures 1 and 2, we ran colony PCR and gel electrophoresis to confirm the presence of our inserted genes. The red boxes in both results highlight the successful transformation of our gene into our two vectors. The bands that are boxed in red match the band in the positive control, therefore confirming our success in transformation.
Localisation
Following, after confirming positive results that our synthesized vectors had our genes, we agroinfiltrated N. Benthamiana. We chose to agroinfiltrate both vectors, pCAMBIA1302 and PGWB2, as pCAMBIA1302 contains GFP which then will be used for localisation. After agroinfiltration, we ran localisation tests to determine if our signaling peptide worked, and whether our gene, PETase, was expressed.
Fig.3 Localisation of PETase in N. benthamiana, showcasing the successful transport of PETase in N benthamina out of the nucleus.
From the localization analysis, we were able to conclude that our target protein was successfully expressed and localised outside of the nucleus, which was what we aimed for. In Figure 3, we can see that in the control, a lot of the portions in the sample is glowing, meaning the protein wasn’t localised anywhere in specific -- just scattered all around the cell, including the nucleus. However, for the sample with our synthesised gene, IsPETase-Amy3S, the visuals shown from the localised results show us that none of our protein was localised near the nucleus of the cell, all were localised in the extracellular space; therefore meaning the expression and localisation of PETase in N. Benthamiana was successful and effective.
HPLC analysis
In addition to showing that our agroinfiltrated N. Benthamiana has successfully expressed and secreted our target protein, PETase, we mixed the leaves’ crude sample with PET particles to test out if the PETase actually works.
Fig.4 Top graph is the standard BHET and bottom graph is the TPA standard
Fig.5 HPLC results for incubating our N.benthamiana crude sample with PET particles under a solvent for 1 week
Fig.6 HPLC results for incubating our N.benthamiana crude sample with PET particles under a solvent for 2 weeks
Fig.7 HPLC results for incubating our N.benthamiana crude sample with PET particles under a solvent for 3 weeks
Comparing figures 5, 6, and 7 with figure 4, the standard, the HPLC results from all weeks 1, 2, and 3 shows that there was no BHET or TPA detected. This was identified by comparing the peaks in the standard sample graphs with the peaks in the results for each week. As BHET and TPA are the intermediate metabolites for PET, therefore the absence of BHET and TPA means the enzyme PETase in our plant, N. Benthamiana is not working properly yet.
However, because in our localisation analysis we were able to identify that our target protein, PETase, has been localised outside of the cell, we know that our protein has been successfully expressed in N. benthamiana and localised in the extracellular space. Therefore showing that PETase can be successfully expressed in plants and localised in areas where PET particles can populate, which is the extracellular space.
Stable Transformation
In addition to transient transformation, we also attempted to perform transgenic transformation, or commonly also known as stable transformation. However we were not able to successfully perform stable transformation as all our shoot cultures went moldy. After multiple attempts in trying to successfully grow a shoot culture, we were confronted with negative results as all the cultures keep on going moldy.
