Our main strategy used for the project had a fairly straightforward approach, increase MS2 infectivity by developing a more efficient lysis protein. The protein is part of what is known as amurins, and affect Gram-negative bacteria, where they disrupt cell-wall biosynthesis. MS2´s, however, produces lysis through a yet-unclear mechanism. We know the end terminal of the protein is involved in the formation of membrane pores that disrupt membrane potential.
We know the lysis process involves the L protein, but also one of the host’s chaperone, DnaJ. In fact, this protein helps the process take place, by stopping the transmembrane part of the L protein from interfering in the binding of its final target. The exact mechanism by which lysis takes place is not fully clear, but the presence of L protein itself is apparently enough to produce lysis, albeit at a lower rate than along with DnaJ.
By finding target mutations that affect the affinity between these two proteins, we could design mutant phages that would have a higher or lower lytic rate, which could be used in bacterial population control, continuous culture, or to help us understand mechanisms of infection escape.
However, in order to better discern potential regions to mutate, using multiple alignment via tools like Clustal-Omega would be helpful, helping to pick out less-conserved regions, which would ideally correlate with their importance in structure and function.
Clustal-Omega alignment of 53 MS2 L-protein homologues showed the fully conserved sites. As stated, we avoided mutating any of these residues because complete conservation suggests they are critical for folding or pore function.
We thus divided the sequence of L-protein between us to test for possible mutations at different points in the structure and their overall impact.
The division was as follows:
1 metrfpqqsqqtpastnrr (1-19)
2 ****rpfkhedypcrrqqrsst (20-38)
3 lyvliflaiflskftnql (39-56)
4 lslleavirtvttlqqllt (57-75)