Auxotrophic Strain Design via Conditional Essentiality and Rescue Analysis

Pipeline for designing auxotrophic E. coli strains: regulatory FBA across 45 carbon sources, single/double/triple knockout screens, and ResMut rescue via a multi-key (EC / BiGG / MetaNetX) reaction library.

Layout

data/        iML1515.xml, iMC1010.csv, delta_gene_rules_normalized_v2.csv, carbon_sources.csv
scripts/     01_rfba_multi_carbon.py → 02_build_reaction_library.py → 03/04/05_*_knockout.py
src/         knockout_utils.py, visualization.py
outputs/     generated CSVs (gitignored)

Install

git clone https://github.com/<user>/auxotroph-design.git && cd auxotroph-design
pip install -r requirements.txt

Python 3.10, COBRApy 0.29, GLPK. Download iML1515.xml from BiGG into data/.

Run

python scripts/01_rfba_multi_carbon.py        
python scripts/02_build_reaction_library.py   
python scripts/03_single_knockout.py
python scripts/04_double_knockout.py
python scripts/05_triple_knockout.py

Each stage reads the previous stage's CSVs from outputs/. Total runtime ~6–10 h on 4 cores (stage 04 dominates).

scripts/rfba_constraints.py runs the rFBA validation on 4 hand-picked conditions; independent of the main pipeline.

Key outputs

File	Contents
multi_carbon_off_reactions.csv	regulatory OFF reactions per substrate
{single,double,triple}_ko_conditionally_essential.csv	CE designs per substrate
{single,double,triple}_ko_rescue_dedup.csv	rescue functions per design

Parameters

Hardcoded at the top of src/knockout_utils.py — these are the values that produced the published figures.