Our team’s goal is to enhance the quality of lumbrokinase enzymes by minimizing intricate production procedures, reducing resource wastage, eliminating unnecessary sacrifices of life, and thereby improving the overall product quality. In our team’s plan, we decided to introduce the IPTG induction to benefit the industrial aspect of lumbrokinase production. With the help of pET-22b with the IPTG induction site, we are able to insert the lumbrokinase gene with the testing process, such as DNA sequencing and large scale restriction enzymatic reaction, to guarantee the functionality and modify it into pET-22b-LK. Furthermore, having the biological methanic of switches; IPTG can release protein with the correct condition while kill-swift can prevent incidents that insure usage safety.
To scale up production, we replicate the vector with the lumbrokinase insertion, resulting in an excess amount of plasmids. Then, we put E. coli colonies into a container with controlled time and control temperature for best production (we will be testing these variables in the future). After having a mass amount of pET-22b-LK vector, we employed a large diameter centrifuge (e.g., scanspeed 1736R) following IPTG induction to separate the supernatant from the pellet. Subsequently, we can extract lumbrokinase protein from the pallet with French Press to break cell membranes for selecting inhibited lumbrokinase. Moreover, we would like to proceed in different trials to maximize the protein we can produce; we will be testing the temperature, humidity and concentration for E. coli BL21 colonies to obtain a faster growth.