Model

For our project, we planned to modify the major capsid protein of our phages to include a petide tag on the surface. The major capsid protein has its main structural elements conserved through almost all species because without it, the phage cannot assemble and transport its genetic information. For this reason it was important to find outward facing areas flexible enough not to compromise the original purpose of the capsid.

After attaining the amino acid sequence for the major capsid protein of the T7 phage from NCBI, we inserted it into both Phyre2, and AlphaFold, protein structure prediction algorithms. Once the site had predicted the folding pattern, we uploaded the file to Chimera. In Chimera you are able to rotate and view a protein aswell as line it up as closely as possible to existing protein structures. By viewing where the protein bowed we were able to determine which side would face out from the phage. We then selected sites without secondary structures such as alpha helixes or beta sheets as possible targets for sequence insertion.

Having identified these sites as shown in pink, we inserted the spytag peptide sequence alongside a flexible linker on either end (for the C terminous, a linker was added only preceeding the sequence) into the major capsid protein amino acid sequence individually at each target. Once we had our new mutant protein sequences, we put them back into ALphaFold and Phyre2 then Chimera to see if the overall shape and structure conserved while outward presenting the peptide.

Once modeled, it became clear that the carboxyl terminous mutation as shown above was our best bet. The main structure looks incredibly similar to the original with the acception that the C terminous extends out past the main body of the protein containing the spytag peptide.

Above is an example of a mutation that we originally thought would work, but too heavily modified the structure of the overall protein. Inserting the spytag within the axial loop domain seems to have added an extra beta sheet and moved the location of structures downstream of the sequence.

Because we were able to test possible mutation sites before ever making a genetic modification to our phages, we saved time in the wet lab that we would have spent making random modifications and testing for viability.