Constructs#
Shell fusions#
The shell protein is expressed with a hexahistidine tag for purification and, in the evolved version, a λN⁺ peptide grafted onto its lumenal surface as the RNA-binding handle.
Two tag architectures are in play, because the first one may be the reason purification has not recovered assembled cages:
| Architecture | Rationale | Status |
|---|---|---|
| His tag via GS linker | Flexible linker, standard practice | Built; suspected of being occluded in the assembled cage |
| Direct terminal His tag | Reported in published supplementary work to assemble successfully | Built as the discriminating test |
See Results for why this matters more than it should.
Reporter constructs#
Several expression constructs carry mScarlet (26.7 kDa) as a fluorescent cargo reporter, so that co-elution of cargo with the shell peak can be followed by eye during size exclusion. Approximate masses used for gel interpretation:
| Species | Mass |
|---|---|
| QtEncapsulin monomer | 32.2 kDa |
| QtEncapsulin + peptide fusion | 34.1 kDa |
| mScarlet | 26.7 kDa |
| Assembled T=4 shell | ~7.7 MDa |
Cargo tags#
The protein handle. dCas9 is fused to the native cargo-loading peptide, whose
core motif is the five residues TVGSL. Five residues is short enough that it is
unlikely to interfere with folding or function — a real consideration for a
protein that has to undergo large conformational changes to load its guide and
find its target.
The RNA handle. A boxB stem-loop is placed in the sgRNA scaffold, where it is caught by the λN⁺ peptide on the shell interior. λN⁺ carries a known lysine-to-arginine substitution that raises boxB affinity roughly threefold, available as a tuning knob if capture proves too weak.
Controls#
The controls are what make the selection interpretable, and several of them are already built.
Non-targeting guide (s002_NT). Defines unrepressed resistance output. Any
cell that survives at a kanamycin dose the non-targeting control cannot tolerate
is doing something other than what we think.
Shell-free. dCas9 and guide present, no encapsulin. Defines the floor — maximal repression, no rescue possible.
Handle-free shell. Encapsulin without the λN graft and dCas9 without the CLP. Distinguishes genuine handle-mediated capture from non-specific sequestration by a large abundant protein.
Single-handle constructs. Shell with λN but dCas9 without CLP, and the converse. These are the controls that resolve the ambiguity the OR gate introduces — they say which arm any observed rescue is running through.