Part I : Behavior Control Lab

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      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. The completion of the behavior control lab both confirmed our hypothesis and provided a qualitative confirmation of the mutation’s successful correction.

Part II : CRISPR

Part III : CRISPR Confirmation (genotypic)

Part IV: Designing of the new constructs

Part V: Gibson Assembly of Constructs

Part VI: Deciding which fluorescent marker to use (water melon tubes, and plates)..deciding then to only use the GFP ones

Part VII: Genotypic and Phenotypic Proof of Concept

• a. Agarose gel electrophoresis of plasmid constructs (agarose gel electorphoresis protocol)
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      On top of the results from the Sialic assay, we wanted 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 has performed it before.
• b. Phenotypic Proof (Transformation and growing on selection plates)

Part VIII: Quantifying Protein

• a. GFP protein purification using column chromatography
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      Our goal was to isolate A1AT secreted by all constructs so the protein can be accurately analysed without obstructions. Some of the proteins produced by constructs containing the histidine tag were purified using the his-tag protein purification with nickel column tubes and others were purified using the standard column chromatography method. We purified the proteins before conducting GFP fluorescence readings, the sialic acid assay (ELISA test), and gel electrophoresis.
• b. Sialic Acid Assay - original version of protocol
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      We decided after finishing the behavior control lab that we should seek quantitative results that will helps us more accurately determine if the gene edit was successful. The sialic acid assay was performed to determine the sialic acid levels secreted by each construct. Based on previous literature, we determined that the unmutated constructs would secrete more sialic acid. On the other hand, 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 unmutated construct, to determine if our gene edit was successful. If successful, the CRISPR edited constructs would show sialic acid secretion similar to that of the unmutated construct.

Part IX: Incorporation of New information to use His-tag

• 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
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      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
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      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 amount of sialic acid secreted. Since our previous sialic assay was unsuccessful due the the extremely complex procedure and lack of conclusive results, the commercial kit version allowed for a direct method to target the sialic acid secreted through a GFP reporter system. 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.
• h. Final Polyacrylamide Gel Confirmation of Constructs
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      Determine whether the CRISPR edited SERPINA1 E342K with point mutation construct was successfully edited by measuring the amount of polymerization that has occured due to the production of mutated 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. Because our originally electrophoresis was not conclusive, due to wrong concentration that we used in the 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.