0. IMPORTANT
0.1 General
0.2 Notation
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 Experimental structures for reference
2.2 AlphaFold model for Cn PhaC1
2.3 Key structural regions (Cn PhaC1 numbering)
2.4 Substrate-binding tunnel residues
2.5 Tunnel geometry notes
3. Dataset Scope, Limitations, and Compensating Strategies
3.1 What your dataset contains
3.2 Implications and honest limitations
3.3 What this dataset IS good for
3.4 Compensating strategies
3.5 Class II reference comparison
4. Experimental Mutation Database
4.1 Key literature to mine for Cn PhaC1 mutations
4.2 Gain-of-function mutations (change in substrate specificity or activity)
4.3 Neutral mutations (no significant effect on specificity)
4.4 Deleterious mutations (loss of activity or expression)
4.5 Combinatorial / double mutants
4.6 Thermostability mutations
4.7 Positions that have NOT been mutated in literature
4.8 Data quality notes
5. Your Starting Enzyme (Wild-Type Cn PhaC1)
5.1 Identity
5.2 Known properties of WT Cn PhaC1
5.3 Full WT sequence
5.4 Substrate-binding pocket region
5.5 Catalytic and key residue positions (for quick reference)
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 Eventual In vivo PHA production conditions
7.3 In vitro activity assay (if used)
7.4 PHA analysis
8. Reasoning Guidelines for LLM
8.1 Dataset context — tell the LLM explicitly at session start
8.2 Prioritization criteria (in order, adjusted for this dataset)
8.3 Required output format for mutation suggestions
8.4 Reasoning I do NOT want
8.5 Especially useful prompts for this dataset type
8.6 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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