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)
• 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

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)
• c. Sialic Acid Purification redone using new protocol (6 trials)
• d. Fluoroscence Microplate Reading to Quantify Protein Concentration of Constructs using GFP standard curve
• e. Fluorescence Microplate Reading to Quantify Sialic Acid Concentration using Sialic Acid Standard Curve
• f. Final Polyacrylamide Gel Confirmation of Constructs