As the initial step of the project, we first designed a primer to transform our synthesised gene from the pCAMBIA1302 vector into the PGWB2 vector (more about vector design can be found in the part collection page).
Following, we used infusion cloning to transform our synthesised gene into the pCAMBIA13021302pCA vector and the PGWB2 vector. To confirm our success in transformation, we grew our bacteria in plates and ran colony PCR and gel electrophoresis to confirm that our inserted gene was present in the plasmids. In doing this, we had to purify our plasmid by using the plasmid extraction kit from Flavorgen. After confirming that our PGWB2 plasmid contained our synthesised gene, we continued to transform it from both the pCAMBIA1302 and PGWB2 vectors into Agrobacterium. Following, we grew the two transformed types of Agrobacterium in plates in preparation for colony PCR and gel electrophoresis. We picked out bacteria colonies from the plates to run colony PCR and gel electrophoresis to confirm that our synthesised gene is present in the Agrobacterium. After that, we inoculated the Agrobacterium for 3 days and agro-infiltrated the Nicotiana benthamiana leaves with the inoculated Agrobacterium -- the plants were left to grow for a week. Next, we freeze-dried the plant samples by using liquid nitrogen and mixed them with PET particles under a solvent. Following, we ran localisation tests on the agroinfiltrated leaves and ran HPLC analysis on the sample mixed with PET particles to test for gene expression and protein functionality.