Team:ASIJ Tokyo/Demonstrate
DEMONSTRATE
Our goal was to prove that the mutated sequence of SERPINA1 has a greater mass, because its polymerization causes aggregates in the cell. We were able to do so through transformation of our constructs in the same test tube to differentiate the mass of the mutated and the unmutated SERPINA1 constructs. 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, and does not polymerize. To confirm our results, this time, we tagged the unmutated SERPINA1 construct with RFP and the mutated SERPINA1 with GFP. 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. The CRISPR tube showed the same result as the unmutated tube, suggesting that the point mutation E342K has been fixed and that it successfully produces and secretes the SERPINA1 gene, coding for the A1AT protein.
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. |