Part I : CRISPR

Part II : CRISPR

Part III : CRISPR Confirmation (genotypic)

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

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)
• Goal:
      To confirm our hypothesis regarding the secretion behaviour of A1AT in cells as it differs between the M allele and the Z allele. The completion of the behavior control lab both confirmed our hypothesis and provided a qualitative confirmation of the mutation’s successful correction.
  Through our research we discovered that A1AT produced by SERPINA1 E342K (ZZ allele) with point mutation gene will polymerize within the cell and not allow it to be secreted whereas the protein produced by the SERPINA1 (MM) will successfully exit the cell into solution. We concluded if each construct was tagged with distinctly different coloured fluorescence reporter we would see a qualitative difference in where the fluorescence was located. If we observed a pattern in the location of the fluorescence across all of our construct which matched the behaviors of the specific construct, we could conclude qualitatively.
• b. Phenotypic Proof (Transformation and growing on selection plates)

Part VIII: Quantifying Protein

• a. GFP protein purification using column chromatography
• 
      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
• Goal:
      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)
• 
      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.
• f. Fluoroscence Microplate Reading to Quantify Protein Concentration of Constructs using GFP standard curve
• 
      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
• h. Final Polyacrylamide Gel Confirmation of Constructs
• 
      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.