Group Final Project

Bacteriophage Engineering

GROUP MEMBERS: Diogo Custodio; Flo Razoux; Katharine Kolin; Mariana Kanbe; Marisa Satsia.

PROJECT MAIN GOAL : Increase the stability of the L protein

GROUP PROPOSAL: We will use the same workflow than in previous HW (e.g. mutagenesis) but adapt it to specific aim(s) based on HW reading material of week 04 (e.g. shorten the L protein to make it not dependant on bacterial chaperone DnaJ anymore).

Please check our most recent updated Google Docs on this.

Here’s a summary of my main individual contributions to the plan for engineering the bacteriophage:

I ran the provided mutational scoring notebook to obtain per-substitution LLR scores for the MS2 L-protein and shortlisted substitutions with positive scores. The full scoring results are included in a table on my Homework 5 page.

I then cross-checked these shortlisted mutations against the provided experimental mutant dataset, L-Protein Mutants, which reports amino acid substitutions and their measured lysis phenotypes.

The overlap between the two data suggests that sequence-based LLR scores capture only part of the functional landscape of the MS2 L-protein. More broadly, positive LLR scores may reflect sequence plausibility or local biochemical compatibility, but they do not fully account for higher-order constraints such as host-factor dependence, membrane behavior, and oligomer formation.

Therefore, I decided to select five candidate mutations by combining positive LLR scores with biological reasoning about the protein’s distinct functional domains, treating LLR scores as a prioritization tool for experimental testing rather than as a direct predictor of lytic function.

The MS2 L-protein is organized into distinct functional domains:

  1. Hydrophilic N-terminal region involved in DnaJ-mediated folding
  2. Transmembrane/C-terminal region responsible for membrane insertion and pore formation

The two soluble-region mutants, S9Q and C29R, were chosen to probe effects on folding and possible DnaJ dependence, whereas the three transmembrane mutants, A45L, T52L, and N53L, were chosen to probe membrane insertion and oligomerization.

Mutant 1 - S9Q (soluble, LLR = 2.014)

Sequence: METRFPQQQQQTPASTNRRRPFKHEDYPCRRQQRSSTLYVLIFLAIFLSKFTNQLLLSLLEAVIRTVTTLQQLLT

Selection Rationale: High positive score in the soluble region (putative DnaJ-interaction domain). Ser→Gln increases hydrogen-bonding potential and may alter surface chemistry without strongly destabilizing the fold.

Mutant 2 - C29R (soluble, LLR = 2.395)

Sequence: METRFPQQSQQTPASTNRRRPFKHEDYPRRRQQRSSTLYVLIFLAIFLSKFTNQLLLSLLEAVIRTVTTLQQLLT

Selection Rationale: One of the strongest positive-scoring substitutions in the soluble region. Adds a positive charge that could reshape chaperone-recognition or interaction surfaces.

Mutant 3 - A45L (TM, LLR = 1.539)

Sequence: METRFPQQSQQTPASTNRRRPFKHEDYPCRRQQRSSTLYVLIFLLIFLSKFTNQLLLSLLEAVIRTVTTLQQLLT

Selection Rationale: Hydrophobic substitution in the transmembrane segment. Ala→Leu increases hydrophobicity and may stabilize membrane helix packing/insertion and oligomer stability.

Mutant 4 - T52L (TM, LLR = 1.814)

Sequence: METRFPQQSQQTPASTNRRRPFKHEDYPCRRQQRSSTLYVLIFLAIFLSKFLNQLLLSLLEAVIRTVTTLQQLLT

Selection Rationale: Polar→hydrophobic change in the TM region. Thr→Leu may increase membrane compatibility and reduce local insertion/misfolding penalties.

Mutant 5 - N53L (TM, LLR = 1.865)

Sequence: METRFPQQSQQTPASTNRRRPFKHEDYPCRRQQRSSTLYVLIFLAIFLSKFTLQLLLSLLEAVIRTVTTLQQLLT

Selection Rationale: Polar→hydrophobic change in the TM region with a strong positive score. Selected as an additional TM-stabilizing candidate.