Emergomyces_constructs_2025

This code is to generate codon-adjusted fluorescent proteins for Emergomyces africanus and Histoplasma capsulatum.

Edward Wallace, [email protected], 2025

Funded by the Wellcome Trust Bioimaging award.

Contents

data

Input data:

• native sequences for codon counts from FungiDB. Precisely, Histoplasma capsulatum or Emergomyces africanus CDSs with annotation GO:0003735 "structural constituent of ribosome". Downloaded by EW in February 2025.
• protein sequences for fluorescent proteins from fpbase.org. Downloaded by EW February 2025.

Red FPs:

• mCardinal
• mCherry-XL
• mCherry
• mKO2, aka mKusabira-Orange2
• mNeptune2.5, but the filenames are called mNeptune2-5 because filenames don't like dots
• mScarlet-I3
• mScarlet3-H, aka mYongHong

Green & yellow FPs:

• mGreenLantern
• mNeonGreen
• fuGFP, aka Free Use GFP
• hfYFP, aka Hyperfolder YFP
• mStayGold, aka QC2-6 FIQ

Cas9, not an FP:

• RdCas9_NLSHA - Cas9 enzyme for eukaryotic CRISPR including 2x NLS and HA tag

designs

Coding sequences for fluorescent proteins, adjusted to codon frequencies of Histoplasma capsulatum ribosomal proteins, moderate GC content, and to avoid select restriction enzyme sites. See source code for details.

src

Source code in python format. Uses the DNAchisel package and Biopython tools.

• codon_adjust_FPs.py - using DNAchisel's DnaOptimizationProblem to design sequences encoding fluorescent proteins
• codon_adjust_Cas9.py - similarly to design for RdCas9_NLSHA, had to relax some optimisation constraints and run for more iterations as a longer harder sequence
• codon_count_functions.py - functions to calculate codon counts and frequencies

How to run the code

To runs the code to design the proteins, from the root directory of of Emergomyces_constructs_2025, in a bash terminal run:

python src/codon_adjust_FPs.py

Computational environment

You'll need the relevant python packages installed. Biopython and DNAchisel, etc.

It should be possible to install the packages using conda:

conda create --name codons2025 --file codons2025_env.txt
conda activate codons2025

Beware, I have not tested this.