The design of our plasmid was illustrated in the graph on the left. Since the conjugated product in our project involves both enzymes and oligonucleotides, our team plan to synthesize enzymes used in this project. Hence, two basic parts were designed, including lignin peroxidase (LiP) [Part ID: BBa_K4837000] and manganese peroxidase (MnP) [Part ID: BBa_K4837001], which were engineered using pSBIK3 vector. These sequences were optimized for E.coli expression.
The promoter used in these two basic parts was arabinose promoter. Purification tag and fluorescent tag were also included for better detection of the optimized enzyme synthesized.
The above biobricks were then designed and modified by azide with an NHS Ester functional group to attach an azide moiety at the 5' of the oligos.
The two azide-modified DNA oligos were then conjugated with the enzyme synthesised by the two plasmids respectively. These two final products were then linked by complementary base pairing of the DNA oligos, forming a "Cascade Enzyme System" of LiP and MnP.
