Team:ASIJ Tokyo/Demonstrate

DEMONSTRATE

Our goal was to prove that the mutated sequence of SERPINA1 has a greater mass, as its tendency to polymerize causes the protein to aggregate in the cell. We were able to do so through the transformation of our mutated and unmutated SERPINA1 constructs (ZZ and MM) in the same test tube to differentiate their mass. For the first trial, we tagged the unmutated SERPINA1 construct with GFP and the mutated SERPINA1 with RFP. As anticipated, the mutated SERPINA1, reported in red, sank to the bottom of the test tube, thus indicating its greater mass. On the other hand, the unmutated SERPINA1 construct, tagged with green, remained at the top. This shows that the unmutated SERPINA1 has a lower mass, as it does not polymerize. To confirm our results, in a second trial we tagged the unmutated SERPINA1 construct with RFP and the mutated SERPINA1 with GFP, thus inverting the constructs fluorescence markers. As expected, the mutated SERPINA1, reported in green, sank to the bottom of the test tube, thus indicating its greater mass. The unmutated SERPINA1, tagged with red, remained at the top. We also tagged the CRISPR construct with GFP and repeated the experiment, combining the CRISPR construct with the mutated SERPINA1 construct. The CRISPR construct showed the same result as the unmutated tube in the same scenario, suggesting that the point mutation E342K has been fixed and that it successfully produces and secretes the SERPINA1 gene, coding for the A1AT protein. This experiment also allowed us to determine that in terms of fluorescence and visibility, GFP was the optimal reporter for our purposes.
Further analysis is needed to determine whether the trapped proteins are trapped inside the endoplasmic reticulum or the cell membrane.

#1: Mutated SERPINA1 tagged with RFP polymerizes and thus sinks to the bottom. Unmutated SERPINA1 tagged with GFP has a lighter mass, so it stays at the top. #2: Mutated SERPINA1 tagged with GFP polymerizes and thus sinks to the bottom. Unmutated SERPINA1 tagged with RFP has a lighter mass, so it stays at the top. #3: CRISPR edited SERPINA1 with no more point mutation tagged with GFP.