Part I CRISPR

Part II CRISPR Confirmation (genotypic)

Part III: Designing of the new constructs (this is just modeling the construct design and not really a protocol)

Part IV: Gibson Assembly of Constructs

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

Part VI: Genotypic and Phenotypic Proof of Concept

• a. Agarose gel electrophoresis of plasmid constructs (agarose gel electorphoresis protocol)
• Goal:
      Determine whether the CRISPR edited SERPINA1 E342K with point mutation construct was successfully edited by measuring the presence 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.
• Why:
      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. 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 VII: Quantifying Protein

• a. GFP protein purification using column chromatography
• b. Sialic Acid Assay - original version of protocol
• Goal:
      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.
• Why:
      We decided after finishing the behaviour control lab that we should seek quantitative results that will helps us more accurately determine if the gene edit was successful. We had theories several methods to obtain quantitative results but in the end decided on the Sialic assay because we already had the materials to perform it.

Part VIII: 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)
• Goal:
      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.
• Why:
      Since our previous sialic assay was unsuccessful due the the extremely complex procedure and lack of conclusive results, the ELISA 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. .
• f. Fluoroscence Microplate Reading to Quantify Protein Concentration of Constructs using GFP standard curve
• g. Fluorescence Microplate Reading to Quantify Sialic Acid Concentration using Sialic Acid Standard Curve
• h. Final Polyacrylamide Gel Confirmation of Constructs
• Goal:
      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.
• Why:
      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. .