To build an optimal biobrick, we came up with 3 different arrangements using the T7 promoter, the osmY tag, the His tag, a single or double terminator, and a GFP reporting system and assembled the parts together using a Gibson Assembly, to then transform in test tubes. The parts chosen were selected on the basis of part literature, taking into account factors such as secretion efficiency and ease of purification. We used the osmY based on its success in past iGEM projects. The his tag was used because it was recommended to us when we went to the Tokyo Tech meetup, part of our integrated human practices, as we learned that the tag optimizes secretion and makes purification easier. For each of the 3 arrangements, we inserted the genetic sequence for the mutated, unmutated, and CRISPR edited SERPINA1 gene.
Promoter-reporter constructs A through C use a double osmY secretion tag and a double terminator system. D through F use a double osmY secretion tag and a single terminator system. G through I use a His and osmY secretion tag with a single terminator system. To test the efficacy of the CRISPR, we used the unmutated SERPINA1 constructs as our positive control, and the point mutated SERPINA1 E342K constructs as our negative control.