Team:ASIJ Tokyo/Results
RESULTS
GFP Fluorescence
By quantifying the secretion of antitrypsin through a GFP reporter system, we determined our optimal construct design. To create a standard curve showing GFP concentration and relative fluorescence intensity, we used concentrations of 0 uM, 8 uM, 16 uM, 32 uM, and 40 uM GFP in 1 mL PBS. Based on this curve, we took our fluorescence readings from our different constructs and used the equation
Relative Fluorescence Intensity=-0.015 + 0.007625(GFP Concentration), with R2 = 0.991927
to determine the concentration of GFP from the fluorescence intensity measured from each construct. The unmutated construct with the his and osmY tag and a single terminator system had the highest relative mean fluorescence of 0.1616 and the highest GFP concentration of 23.1607 uM. To further prove that the construct with the his and osmY tag was the most effective, the data shows that the CRISPR gene edited construct had a relative mean fluorescence intensity of 0.1221 and a GFP concentration of 17.9803 uM, which is higher than the unmutated constructs with double osmY tags with single and double terminator systems, which only had relative fluorescence intensities of 0.0423 and 0.0383 and GFP concentrations of 7.51475 uM and 6.99016 uM, respectively. Thus, looking into future projects using unmutated and CRISPR edited sequences of SERPINA1, a construct designed with a his and osmY secretion tag with a single terminator system will promise optimal secretion, as indicated in these fluorescent readings.
Sialic Acid
The efficacy of our constructs were also quantified through an ELISA, in which we linked a fluorescence antibody to sialic acid, to directly measure the amount secreted by each construct. Sialic acid linked to GFP with concentrations of 0 uM, 10 uM, 20 uM, 30 uM, 40 uM, 60 uM, 80 uM, and 100 uM were used to create a standard curve with equation
Relative Fluorescence Intensity= -0.006725 + 0.060599(Sialic Acid Concentration), with R2 = 0.998676
to determine the concentration of sialic acid from the fluorescence intensity measured from each construct. The CRISPR construct with double osmY secretion tags and double terminators had 100% of the sialic acid levels of the unmutated construct, indicating its efficacy in correcting the point mutation, and secreting expected sialic acid levels. However, its overall low relative fluorescence intensity at 0.620211667 and concentration of 10.3456667 uM reveals the double osmY and double terminator constructs to be ineffective, and thus may not promise optimal results in future experiments, regardless of the success of the CRISPR gene edit in this construct. The constructs with the double osmY secretion tag and a single terminator showed the CRISPR edited construct to secrete 75.4376% of the amount of sialic acid secreted by the unmutated variant, showing success of the CRISPR gene edit. Similarly, the constructs with the his and osmY tag with a single terminator showed the CRISPR edited construct to secrete 74.934689% of the amount of sialic acid secreted by the unmutated variant, showing success of the CRISPR gene edit as well. The relative sialic acid concentration secreted by the unmutated and CRISPR gene edited constructs with the his and osmY tag with a single terminator was 80.9585 uM and 60.666 uM respectively, whereas the unmutated and CRISPR gene edited constructs with a double osmY tag and a single terminator showed sialic acid concentrations of 51.074 uM and 38.529 uM, respectively. Because the percentage of sialic acid secreted by the construct with the his and osmY tag with a single terminator, 74.934689%, and by the construct with the double osmY tag and a single terminator, 75.4376%, was similar, we can use the sialic acid concentrations in determining which construct would demonstrate overall efficacy. In doing so, we can conclude that the construct with the his and osmY tag with a single terminator maximizes sialic acid secretion of both the unmutated and CRISPR edited construct. In addition, we can confirm the polymerization of the protein in the mutated constructs, as sialic acid levels of the mutated construct as a percentage of the unmutated construct remained low at 2.9548545%, 3.3843051%, and 9.7961919% for the double osmY tag and double terminator system, double osmY tag and single terminator system, and his and osmY tag and single terminator system, respectively.
Electrophoresis
- Mutated SERPINA1 with double osmY and double terminator
- Unmutated SERPINA1 with double osmY and double terminator
- CRISPR gene edited SERPINA1 with double osmY and double terminator
- Mutated SERPINA1 with double osmY and single terminator
- Unmutated SERPINA1 with double osmY and single terminator
- CRISPR gene edited SERPINA1 with double osmY and single terminator
- Mutated SERPINA1 with his and osmY tags and single terminator
- Unmutated SERPINA1 with his and osmY tags and single terminator
- CRISPR gene edited SERPINA1 with his and osmY tags and single terminator
In the page electrophoresis stainings, we can confirm that the mutated SERPINA1 constructs in wells 1, 4, and 7 with denser bands around 40 kDa indicate its tendency to polymerize. The unmutated SERPINA1 constructs in wells show bands ranging from 30 kDa to 60 kDa. The CRISPR edited SERPINA1 in wells 3, 6, and 9 show the similar dense bands expressed in the mutant variants, of 40 kDa, however also do indicate small levels of protein at weights of 60 kDa, 50 kDa, and 30 kDa, which are also observed in the unmutated variants of SERPINA1. Because the CRISPR constructs show hybrid characteristics of both the bands from the unmutated and mutated constructs, we may have created an MZ mutant variant. Regardless, because the gel electrophoresis indicated the unmutated, mutated, and CRISPR edited variants to separate differently in the gel, the results remain conclusive and consistent with our other experiments.