Efficiency in Protein Expression with Gibson Assembly in Metacaspases

Metacaspases are cysteine proteases found in fungi, protozoa, and plants. They play a role in programmed cell death, stress response, and protein homeostasis. Protein production can be influenced by inducers that initiate gene expression and expression vectors that contain regulatory elements, enhancing protein synthesis. Currently, protein expression of metacaspases involves a plasmid with a non-inducible promoter. Non-inducible plasmids have promoters continuously active or continuously repressed, resulting in continuous or inhibited production of protein, while inducible plasmids have promoters that can be activated or repressed. Because the metacaspase protein might be toxic, and in order to improve the expression of the metacaspase, we wanted to use a plasmid with an inducible promoter. We successfully joined the inducible plasmid sequence with the DNA sequence coding for the metacaspase through Gibson Assembly, a cloning method that allows the joining of several DNA fragments. The lac operon in the inducible plasmid allows transcription to occur in the presence of lactose. We will present results comparing the production of metacaspase protein with an inducible plasmid versus a non-inducible plasmid to measure the efficiency of protein expression in this new system.