Group Final Project

Plan

Bacteriophages -> L to lyse bacterial cells

  • Release newly produced phages
  • Modification to make more effective for lysing the protein
  • Higher stability for L protein

L protein interacts with host (cellular missionary) with chaperone, DnaJ

  • Influence protein folding
  • POSSIBLE DIRECTION: modifying residues to influence how L protein interacts with host proteins
  • Specific minor acid residues can affect lysis

GOALS

  • Increase stability of L protein
  • Higher titers
  • Higher toxicity of lysis protein

COMPUTATIONAL DIRECTIONS

  • Attempt to improve L stability
    • Computational protein design tools
    • Investigate mutations that could impact protein and host factors interactions

Pull from database what is considered more stable from similar proteins

  • See what is conserved between them
  • What does stability mean in this context? Only in cytosol? Ultimate method of delivery?
    • Use same tool for Week 5
    • Host cell in unfolded, folded by chaperone
  • POSSIBLE DIRECTION: If the efficacy of L protein requires chaperone (working within specific temp range), could we mutate to work in greater temperature range for that chaperone
    • Limit denaturation?
    • Binding (30-37 C) -> will not be able to lyse bacteria

READING (BEFORE NEXT MEETING)

  • Stability, which parts of protein to stability
  • Binding characteristics
  • What we can change, play with

FINAL PROPOSAL:

Bacteriophage Final Project Proposal: Engineering the MS2 L Protein

Brainstorm Session & Chosen Goal

  • Goal: Higher toxicity of lysis protein (Hard).
  • Focus: We aim to engineer the MS2 bacteriophage L protein to induce faster and more severe bacterial lysis by severing its dependence on the E. coli host chaperone DnaJ. Naturally occurring Lodj (L overcomes DnaJ) mutants, which lack the highly basic N-terminal half of the protein, can bypass DnaJ and lyse cells approximately 20 minutes earlier than the wild-type. We will computationally design novel N-terminal variants while preserving the essential C-terminal transmembrane domain and the critical LS dipeptide motif (Leu48-Ser49), which forms the core of its heterotypic protein-protein interactions.

Tools and Approaches

  • In Silico Mutagenesis (Evo Models): We will use advanced genome language models (Evo 1 and Evo 2) to perform in silico mutagenesis. We aim to generate a diverse library of L gene variants with altered or truncated N-terminal domains to bypass DnaJ dependency.
  • AlphaFold 3: We will utilize AlphaFold 3 to predict the 3D structures and biomolecular interactions of our newly generated L protein candidates.

Rationale for Chosen Tools

  • Evo Models: Genome language models like Evo have been trained on vast datasets encompassing over two million bacteriophage genomes. They have demonstrated the unique ability to learn complex evolutionary rules and generate biologically realistic, functional phage sequences with high novelty.
  • AlphaFold 3: AlphaFold 3 provides highly accurate structure predictions for biomolecular interactions. We will use it as a structural filter to ensure that our mutations do not disrupt the critical C-terminal helical domain and that the engineered proteins can still form the high-order oligomeric assemblies in the membrane required for cell lysis.

Potential Pitfalls

  • Endotoxin Release (Manufacturing & Safety): Accelerating the lysis process could lead to a massive, rapid release of bacterial components, including endotoxins and DNA, which poses a significant manufacturing and clinical safety hurdle. These toxins must be rigorously purified before administration.
  • Phage Resistance: Bacteria are involved in a constant evolutionary arms race and could rapidly develop resistance to our engineered, highly toxic phages.
  • Unknown Host Target: While we know the L protein interacts with DnaJ, its ultimate membrane protein target responsible for executing lysis remains completely unknown. Consequently, we cannot use AlphaFold 3 to explicitly optimize the binding affinity between our engineered L protein and its final target.

Pipeline Schematic

  1. Sequence Generation: Input the wild-type MS2 L sequence into the Evo genomic language models to generate a diverse library of variants focused on N-terminal modifications.
  2. Structural Filtering: Run mutant sequences through AlphaFold 3 to evaluate homomeric stability and ensure the vital C-terminal LS motif remains structurally intact.
  3. Initial Plasmid Screen & Measurable Output: To accurately measure lysis without the confounding variables of a full viral infection cycle, we will clone our Evo-generated L library into an arabinose-inducible pBAD vector, such as pBAD24 or pBAD33. We will transform these plasmids into an E. coli K-12 strain (e.g., TB28) to ensure standard host chaperone interactions. After inducing expression with arabinose, we will perform high-throughput growth inhibition assays in liquid culture by continuously tracking OD 600 in 96-well plates. Our primary measurable output for success is accelerated lysis kinetics. To definitively quantify “higher toxicity” we will compute the numerical derivative of OD 600 over time to extract three specific kinetic parameters compared to the wild-type MS2:
    • Maximum rate of decline (−Δ*OD/*600/min ): A steeper decline directly indicates a more potent/toxic lysis protein.
    • Time to minimum population density (minutes): Proves if our Lodj variants induce an earlier onset of lysis.
    • Depth of lysis: The absolute lowest OD 600 value reached.
  4. Whole-Phage Validation: Once we identify the top-performing, highly toxic L candidates from the plasmid screen, we will synthesize those specific genes as DNA fragments and assemble the recombinant full MS2 phage genomes in vitro using multi-fragment Gibson assembly techniques. This will ensure that our engineered L proteins remain fully functional in the context of a complete phage and do not inadvertently abolish viral viability.

References