1) Optional design considerations based on the MS2 genome: Remember the genome structure of the MS2 phage?
Source: [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5446614/](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5446614/)
Notice how the gene encoding the L-protein overlaps with the coat gene and the rep protein? Although the coat (coat proteins) and rep genes (phage replicase) overlap with the L-gene (lysis protein), they are encoded in a different Open Reading Frame (ORF), meaning that the codon read-out is different although they share the same nucleotides. An overview of this is visualized in this Benchling snapshot:
For your protein design you might want to consider the genomic context when deciding on a mutation candidate: Will your mutation in the lysis protein gene result in a different amino acid residue in one of the overlapping genes, too?
If targeting residues of which codons overlap with the codons of other MS2 genes, you might consider creating models of the affected protein as well, and seeing whether their structure will be significantly changed.
You might want to try choosing residues with codons that lead to synonymous mutations in the overlapping genes, or to residues that have similar biochemical properties (e.g avoid replacing a small amino acid with a bulky etc.).
This is an optional consideration to make, because we still have to test whether it is possible to create modified and infective MS2 particles in our upcoming proof-of-concept experiments in the first place. Therefore, you might create some designs that consider the MS2 genome as whole and some designs that ignore the MS2 genome context and optimize the lysis protein as a stand alone protein. It’s up to you!
2) Optional design considerations based on literature
You might notice that the BLAST results show many entries from the MS2 phage itself and of MS2 phage sequences encoded on vectors. Although many of these sequences are not from other species, you might still consider them to identify residues that are variable and are thus a good bet for residue modification.
If you research the taxonomy of the few entries of genomes of other phages, you will find that many of them are derivatives of the MS2 Phage.
However, because many of the few phages displayed in the results are derivatives of the MS2 phage, the sequence could appear more conserved than it actually is.
The reason for why you find barely any sequence similarity to other non-MS2 derived is that the MS2 lysis protein is very unique.
Scientists have in fact identified only few other phages that carry a MS2 “L-(protein)-like” protein such as Hgal1, Ku, PRR1, PP7, which despite little sequence similarity show structural similarity, and in another study, the AVE017 phage – with a sequence similarity to the MS2 phage of only around 38%. You might consider the respective papers and the featured phages to further inform your mutation choices.