Individual Final Project

Goal Demonstrate that you can apply HTGAA concepts (e.g., Benchling workflows, computational protein design) in a focused, well-documented project.

Timetable (work log)

To-do notes

• Keep this page in sync with your slides if the idea changes.

SECTION 1 — Abstract

Ischemia-reperfusion (I/R) injury, where blood supply restoration paradoxically worsens tissue damage, is a critical challenge in conditions like heart attacks and organ transplantation. This project focuses on addressing a specific mitochondrial disruption central to I/R injury: the Cyclophilin D (CypD)-mediated opening of the mitochondrial permeability transition pore (mPTP).

The significance lies in pioneering a targeted therapeutic, as no FDA-approved drugs currently address this precise mechanism. The broad objective is to first computationally design and then experimentally validate a peptide inhibitor of CypD. The central hypothesis is that a peptide, designed using computational tools to target a specific sequence on CypD, can prevent mPTP opening, thereby mitigating calcium overload, ATP depletion, and subsequent cell death.

The first specific aim (HTGAA focus) involves identifying the CypD target and computationally designing the therapeutic peptide, followed by initiating its synthesis and preliminary in vitro validation.

Subsequent aims (Master’s thesis) will involve broader in silico, in vitro (e.g., MTT assay), and in vivo evaluations of the peptide, with a long-term aim of progressing successful candidates through clinical trials to become an approved treatment for myocardial infarction and similar I/R-related conditions.

SECTION 2 — Project Aims

• Aim 1 (HTGAA scope): Identify a mitochondrial dysfunction implicated in I/R injury and computationally design a peptide-based therapeutic to address it. Use bioinformatics databases to select a target (e.g., Cyclophilin D and its role in mPTP opening) and protein-design tools (AI-assisted approaches) to generate a candidate inhibitory peptide sequence. Begin synthesis planning and initial in vitro validation (e.g., cloning into an expression vector such as pET-28a(+), expression in E. coli, and an initial cell-viability assay like MTT).
• Aim 2 (thesis scope): Perform comprehensive evaluation of the designed peptide and explore broader therapeutic pathways related to the targeted mitochondrial disruption—in silico docking/MD of peptide–CypD interaction; in vitro characterization (e.g., assays probing mPTP, ATP levels); progress to in vivo studies for therapeutic potential and safety.
• Aim 3 (impact & iteration): Define criteria to select top peptide candidates, iterate designs (e.g., point mutations, length/chemistry variants), and document decision points for potential translational follow-up.

Notes on tooling (search spaces)

When scouting tools and prior work, look across:

• Literature search portals (PubMed/Nature journals)
• GitHub (open-source repos, pipelines)
• Hugging Face (protein/antibody design models)
• Model families you tried/plan to try: AfDesign, EvoBind, BindCraft, RFdiffusion (record versions/seeds/params)

Deliverables (for this page)

• A clear abstract and three aims (updated as the project evolves)
• A timetable (work log)
• Links to slides and any relevant external resources
• Screenshots/figures you generate during design & evaluation (add as you go)

Group Final Project

Team & credit
Team members: Alireza Hekmati and Yousif Graytee (Baghdad, Iraq).
Yousif is affiliated with the Designer Cells Lab at Yonsei University (Incheon, South Korea). Page text credit: Yousif Graytee. :contentReference[oaicite:0]{index=0}
Lab site: https://designercells.yonsei.ac.kr

Our goal

Make lysis independent of DnaJ by separating the C-terminal from the N-terminal of the MS2 L-protein. :contentReference[oaicite:1]{index=1}

Core idea (high level)

The L-protein’s productive interaction is believed to depend on DnaJ. Its N-terminal is highly soluble and has been reported dispensable for function, while the functional, transmembrane region resides toward the C-terminal. The MS2 lys gene substantially overlaps with the coat protein (cp) and replicase (rep) genes, but a 33-nt (11-codon) window exists with no overlap. :contentReference[oaicite:2]{index=2}

Figures (from our analysis)

1. Genome context & overlaps (Benchling view)
   :contentReference[oaicite:3]{index=3}

2. Non-overlapping 33-nt region highlighted
   :contentReference[oaicite:4]{index=4}

3. N-terminal extent vs. overlaps
   :contentReference[oaicite:5]{index=5}

4. C-terminal/replicase overlap context
   :contentReference[oaicite:6]{index=6}

5. Zoom on the non-overlapping window
   :contentReference[oaicite:7]{index=7}

Explanation (from our notes)

• The lys coding region overlaps with both cp and rep genes for most of its length. :contentReference[oaicite:8]{index=8}
• There is a 33-nt region that does not overlap any other gene and maps to part of the N-terminal (not the functional C-terminal transmembrane domain). :contentReference[oaicite:9]{index=9}
• Idea: introduce a stop codon within that non-overlapping window so the N-terminal is translated separately from the C-terminal segment, avoiding changes to cp/rep while allowing the C-terminal to function independently for lysis. :contentReference[oaicite:10]{index=10}
• This mirrors prior observations that C-terminal truncations can be sufficient for lysis; here, rather than deleting the N-terminal, it is separated to minimize effects on overlapping genes. Hypothesis: DnaJ-dependence and steric hindrance relate to the N-terminal; separating termini could let the C-terminal act without DnaJ. :contentReference[oaicite:11]{index=11}

What we plan to document next

• Rationale and design sketches (already above).
• In-silico checks & controls (non-overlapping region annotations, reading-frame sanity).
• Literature pointers on MS2 L-protein function and DnaJ dependence (to be added as we curate references).
• Evaluation plan outline (conceptual only).