Team:ASIJ Tokyo/Experiments

To confirm our hypothesis regarding the secretion behavior of A1AT in cells as it differs between the M allele and ZZ allele we conducted a behavior control lab. Through our research, we discovered that antitrypsin of the SERPINA1 E342K (ZZ allele) with point mutation variant polymerizes within the cell, thus resulting in a greater cell mass as the protein is unable to be excreted. A protein of the SERPINA1 (MM) variant will successfully exit the cell, therefore resulting in lesser cell mass. To observe this behavior in our own scenario, each construct was tagged with a distinctly different colored fluorescence reporter, enabling us to see a qualitative difference in where the fluorescence, and therefore the protein, was located. In our experiment, the pattern in the location of fluorescence, due to the mass of the cells, followed the expected behavior. We also tried different fluorescence markers and compared the scenarios of an unmutated protein and a protein we conducted CRISPR on. The completion of the behavior control lab confirmed our hypothesis, provided a qualitative confirmation of the mutation’s successful correction, and decided the fluorescence marker used throughout the rest of the expirement..

a. Agarose gel electrophoresis of plasmid constructs (agarose gel electorphoresis protocol)

We conducted the electrophoresis as a way to collect more evidence to establish that the CRISPR edited SERPINA1 E342K with point mutation construct was edited successfully and determine if the proteins produced behaved like the unmutated A1AT. The mutated construct, prone to polymerization is expected to have a larger weight, and thus travel less in the gel. On the other hand, the unmutated construct, with no polymerization, is expected to have a lighter molecular weight and thus travel farther in the gel. This method was chosen because the materials were readily available to us and many members of the team have performed it before.

Our goal was to isolate A1AT secreted by all constructs so the protein can be accurately analyzed without obstructions. Two methods of purification, as well as several protein tags, were tested out. The two methods or column chromatography were His-tag specific protein purification with nickel column tubes and others were purified using the standard column chromatography method. Utilizing the His-tag was a major player in our success. We purified the proteins before conducting GFP fluorescence readings, the sialic acid assay (ELISA test), and gel electrophoresis.

The sialic acid content of a cell is a major characteristic differentiating mutated and unmutated variants. Therefore we chose to conduct a sialic acid assay as a means by which to attain quantitative results to determine with greater accuracy if the gene edit was successful. Based on previous literature, we determined that the unmutated constructs would secrete more sialic acid. The mutated constructs were expected to secrete less, due to its tendency to polymerize in the cell, preventing its ability to exit the cell. This assay would allow us to determine the sialic acid levels for each construct and thus allow us to compare the CRISPR edited construct with the unmutated construct and determine based on the proximity of the sialic acid contents if our gene edit was successful. If successful, the CRISPR edited constructs would show sialic acid secretion similar to that of the unmutated construct.

a. Redesign construct to include His-tag
b. Gibson assembly
c. Transformation
d. Protein Purification using His Tag Protein Chromatography - redone Oct 10 (10 trials)
e. Sialic Acid Purification redone using new protocol (6 trials)
f. Fluoroscence Microplate Reading to Quantify Protein Concentration of Constructs using GFP standard curve

In another attempt to determine the optimal construct and determine whether the mutation was successfully corrected, we quantified the secretion of A1AT through a GFP reporter system. 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.

g. Fluorescence Microplate Reading to Quantify Sialic Acid Concentration using Sialic Acid Standard Curve

Since our previous sialic assay was unsuccessful due the extremely complex procedure and lack of conclusive results, we decided to conduct a commercial kit version. A sialic acid assay provides for a direct method to target the sialic acid secreted through a GFP reporter system. Our goal was to quantify the amount of sialic acid secreted by each construct. By doing so, we could compare the CRISPR edited construct with our unmutated construct to determine if our gene edit was successful. The mutated constructs would serve as a negative control, as its tendency to polymerize would result in the least sialic acid secretion. By using an antibody tagged with GFP, we were able to directly target the sialic acid secreted and thus quantify the fluorescence, which was directly correlated to the amount of sialic acid secreted. And, as expected, after 10 reading of each construct, we were able to find the average fluorescence intensity and thus determine the standard error for each, to conclude that our results were statistically significant.

Because our original electrophoresis was not conclusive, due to a wrong concentration used in gel, we decided to take on a 12% SDS Page Electrophoresis. This would allow us to compare the CRISPR constructs with the unmutated constructs to determine if our gene edit was successful. We once again expect to see a mutated variant with a much greater mass than the unmutated variant and ideally a genetically modified variant similar to an unmutated variant.

EXPERIMENTS