1. Enzyme Family Background
1.1 Classification
1.2 Reaction chemistry
1.3 Substrate scope terminology
1.4 Why substrate specificity is structurally interesting
2. Structural Information
2.1 Available experimental structures
2.2 AlphaFold models
2.3 Key structural regions
2.4 Known substrate-contacting / selectivity residues
2.5 Tunnel geometry notes
3. Sequence Dataset Summary
3.1 Dataset composition
3.2 Taxonomic distribution
3.3 Alignment properties
3.4 Top mutual information positions
4. Experimental Mutation Database
4.1 Key literature to mine
4.2 Gain-of-function mutations (toward mcl/broader specificity)
4.3 Loss-of-function / specificity-narrowing mutations
4.4 Combinatorial / double mutants
4.5 Thermostability mutations
4.6 Notes on data quality and comparability
5. Your Starting Enzyme (Wild-Type)
5.1 Identity
5.2 Known properties
5.3 Sequence — full
5.4 Sequence — substrate-binding pocket region
5.5 Alignment position mapping
6. Engineering Target
6.1 Primary goal
6.2 Secondary goals
6.3 Acceptable tradeoffs
6.4 Hard constraints — DO NOT VIOLATE
6.5 What has already been tested
7. Production and Assay Context
7.1 Expression system
7.2 PHA production conditions
7.3 Analytical method
8. Reasoning Guidelines for LLM
8.1 Prioritization criteria (in order)
8.2 Required output format for mutation suggestions
8.3 Reasoning I do NOT want
8.4 When hypotheses conflict
8.5 My background
9. Session Log
Session [DATE]
Session [DATE]
10. Experimental Results Log
Experiment [DATE / ID]
JESSEE SVOBODA — HTGAA Spring 2026
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