Mindmap mindmap root((Natural Indigo - bacterially derived)) Audience - Funding and Implementation Lab spaces + backing → fibersheds Community biolabs Indirect pulls from DBs; NCBI, UniProt, KEGG, Reactome AI protein/peptide design PepMLM from HuggingFace for peptide generation Structure prediction + docking/MD Binder design: AfDesign / RFdiffusion / EvoBind / BindCraft Automation & coding Google Colab protocols → Opentrons robots Scale experiments, iterate fast Protein Science & Design Amino acids & stereochemistry L vs D amino acids, helix handedness Secondary structure α-helices & variants, β-sheets → aggregation Amyloids as stable materials Model protein eGFP: primary→tertiary structure characterization Genetic Engineering Methods DNA read/write/edit basics Gel electrophoresis, Benchling workflows PCR chemistry Phusion HF Master Mix components/purpose Assembly Gibson Assembly Cloning & expression Vector design → E. coli expression Regulatory Logic & Circuits Post-transcriptional vs post-translational control Endoribonucleases for RNA cleavage vs proteases for protein degradation Cell-Free Systems Why cell-free? Speed - no culturing! Open control of DNA, amino acids, energy, enzymes Toxic proteins OK; supports UAAs Bioproduction & Pathways Carotenoids in E. coli crtE + crtI + crtB → lycopene + crtY → β-carotene Automation mindset What new questions when everything is automated? Phage Engineering: MS2 Lysis protein L N- vs C-terminal roles; C-term TM drives lysis 33-nt non-overlapping window Introduce stop codon to split N from C → DnaJ-independent lysis Preserve overlaps with cp/rep genes Mitochondrial Therapeutics Target: Cyclophilin D “CypD” Block mPTP opening in I/R injury Design peptide inhibitor; plan in vitro/in vivo validation Knowledge Sources NCBI GenBank • UniProt • KEGG • Reactome