To achieve assembly of the protein tip complex, there were several goals that had to be accomplished:

1. THE BIOLOGICAL BLUEPRINT: FIND OUT HOW NATURE BUILDS THE TIP COMPLEX
• Understand the current models of T3SS needle tip assembly
• Define the physical parameters of the needle filament and the needle tip complex
• Determine the solution structure of tip proteins
• Establish own model based on current evidences


2. DESIGN AND BUILD A DNA NANOSTRUCTURE TO MIMIC NATURE'S CONSTRUCTION SCAFFOLD
• Design a range of DNA scaffolds that mimic the natural needle template
• Obtain the sequences for the DNA strands necessary to construct the scaffolds
• Perform DNA origami to construct the DNA scaffolds
• Demonstrate assembly of the barrel template through atomic force microscopy (AFM)


3. OBTAIN THE MATERIALS NEEDED TO ASSEMBLE THE T3SS TIP COMPLEX
• Generate expression vectors containing our tip protein sequence
• Clone these vectors into T7 E. coli for expressing and harvesting our tip proteins
• Purify our tip proteins via histidine affinity chromatography and size exclusion chromatography
• Label our purified protein with Alexa488 dye for single molecule fluorescent microscopy


4. DEVISE CHEMISTRY CONJUGATION METHODS TO ATTACH THE TIP PROTEINS ONTO OUR DNA SCAFFOLD
• Investigate a range of conjugation methods for attaching proteins to DNA
• Produce and purify tris-NTA DNA for use with different scaffolds


5. VISUALISE THE ASSEMBLY OF THE T3SS TIP COMPLEX (KINETICS + SINGLE MOLECULE FLUORESCENCE)
• Kinetics
• Optimisation of biolayer interferometry protocols for DNA-DNA and DNA-protein interactions
• Characterise the kinetic values of 10bp NTA DNA to monomer template
• Characterise the kinetic values of single protein to monomer template
• Assemble the protein tip using DNA racquet template
• Characterise the kinetic values of five proteins to racquet template
• Determine if protein complex assembles cooperatively
• Single Molecule Fluorescence
• Characterise the background intensity of neutravidin surface
• Label tip proteins with fluorophore for single molecule microscopy
• Characterise the intensity of a single fluorophore
• Characterise the non-specific interaction of fluorophore with neutravidin surface
• Observe the intensity vs. time profile of fluorophore at the presence of our DNA scaffold

Check out our awesome workflow here!