Design Experiments From Reported Results

 Long Analysis Plan Copied

Evidence Basis for the Domain-Function Rescue Design

LCI9 localizes to the pyrenoid starch-sheath interface, and lci9-2 mutants show disrupted starch plates, reduced Ci affinity, and slower growth under very low CO₂; complementation with native-promoter LCI9 restores function, establishing the rescue framework for domain dissection LCI9 pyrenoid starch sheath study.

Precedent from other CBM systems supports the mutation strategy: CBM-like domain truncation in Amuc_1547 abolished carbohydrate sensing without abolishing the catalytic domain Amuc_1547 CBM-like domain function, and point mutations at a non-catalytic carbohydrate-binding site (Y380A) sharply reduced catalytic efficiency ~10-fold without gross structural loss Barley alpha-amylase sugar tongs mutagenesis. CBM deletion can also decouple domain stability/function, as in supercharged CBM2a-Cel6B where CBM changes altered activity without breaking the catalytic domain Supercharged CBM cellulase domain design.

Figure is a hypothesis scaffold, not data: the only observed anchor is that native LCI9 fully rescues lci9-2 (sheath circularity, Ci affinity, low-CO₂ growth) LCI9 complementation rescue data.

Experimental Plan

1. Constructs: Clone LCI9 with native promoter into the lci9-2 background: (a) \u0394CBM20 deletion; (b) \u0394coiled-coil deletion; (c) CBM20 aromatic/stacking residue alanine mutants (CBM20 binding typically uses W/Y stacking; select residues from AlphaFold-Multimer v2 model and CBM20 alignments, as the source study used AF-Multimer v2 and MEGA11 LCI9 domain annotation workflow). Add mNeonGreen tag, verify expression by immunoblot and localization by confocal with STA2-mCherry.
2. Controls: WT CC-4533, uncomplemented lci9-2, lci9-2:LCI9 (full rescue), plus \u0394CBM20 expression-level-matched control; verify lci9-2 is not a partial knockout (truncated protein noted as a limitation lci9-2 partial knockout caveat).
3. Readouts: (a) sheath circularity and starch-plate morphology by confocal + SEM (200 granules/genotype in source work; n\u22654 biological replicates); (b) plate clashes, n\u226530 cells/genotype, \u22653 independent transformant lines each; (c) Ci-affinity via Clark-type O₂ electrode, fit K_0.5 and V_max by Michaelis-Menten ( Ci-affinity kinetics method in source study Ci affinity electrode assay); (d) growth curves under <0.01% CO₂.
4. Analysis pipeline: Python (scipy curve_fit for K_0.5/V_max with bootstrap CIs; statsmodels ANOVA with post-hoc Tukey; scikit-image circularity quantification; seaborn/Plotly visualization); pre-register expected effect directions per construct.

Interpretive Logic and Blind Spots

Expected pattern if CBM20 binding is essential: \u0394CBM20 and binding-site mutants phenocopy lci9-2; \u0394coiled-coil may show partial rescue if the coil only positions the CBM. Counter-interpretation: deletions may misfold or mislocalize \u2014 localization imaging must precede phenotype calls. Known unknowns: exact CBM20 ligand preference of LCI9 (amylose vs amylopectin) is untested; AMY3 CBM45 data show CBMs can also mediate dimerization independent of binding specificity AMY3 CBM45 dimerization evidence \u2014 so a \u0394coiled-coil phenotype could reflect oligomerization, not carbohydrate binding. Disproof of CBM-necessity: full rescue by \u0394CBM20 or by binding-site mutants with intact localization.