Group Final Project

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Group Brainstorm on Bacteriophage Engineering

Proposal by: Sameen Nasar (London, Lifefabs), Robert C Beck (London, Lifefabs) to be joined by Jean Colmenares (Lima, Peru)

As per instructions of Node leaders & TAs, this will be put on hold, hopefully we will get to do this over the summer

Group Project Goal:

Engineering a chaperone-independent efficient MS2 lysis protein

Project Rationale:

The efficacy of bacteriophage MS2 as an antibacterial agent is currently limited by the host’s ability to evolve resistance. Specifically, E. coli can mutate the molecular chaperone DnaJ (e.g., at position P330), disrupting the essential interaction required for the MS2 lysis (L) protein to fold and function [1.] This interaction is required for proper function of the lysis protein, as DnaJ binds to the N-terminal domain of MS2 lysis protein and alleviates its inhibitory effect on lytic activity.

We propose engineering a self-activating L protein by replacing its inhibitory, chaperone-dependent N-terminal region with a computationally designed, thermodynamically stable scaffold. As this original domain is dispensable for actual lysis but creates the DnaJ dependency [2], our redesign conceptually eliminates the need for the molecular “handshake” between host and phage, allowing MS2 to fold independently and bypass bacterial control mechanisms entirely.

Schematic

MS2 Protein & DnaJ Sequences
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AlphaFold-Multimer
Map the DnaJ binding interface

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RFDiffusion
Design a stable, independent N-terminal scaffold

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ProteinMPNN
Generate amino acid sequences for the new scaffold

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ESMFold
Confirm the new single-chain mutant folds correctly

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AlphaFold-Multimer
Confirm the mutant no longer binds to DnaJ
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Final L Protein Mutant for Synthesis

References

  1. Chamakura KR, Tran JS, Young R. MS2 lysis of Escherichia coli depends on host chaperone DnaJ. J Bacteriol. 2017;199(9):e00058-17. doi:10.1128/JB.00058-17.

  2. Chamakura KR, Edwards GB, Young R. Mutational analysis of the MS2 lysis protein L. Microbiology (Reading). 2017;163(7):961–969. doi:10.1099/mic.0.000485.