Data#

Fig S1. Schematic overview of the creT-based positive selection strategy in a repressed (a) and an activated (b) state.

Fig S2. KanR-based sgRNA sequence including the spacer and the boxB insertion sites.

Fig S4. Analytical SEC of QtEnc-His across four purification workflows, comparing the starting fraction against the precipitation step on a Superose 6 10/300 GL column. The clarified lysate supernatant (a, c) and the resuspended lysate pellet (b, d) were each carried forward by heat precipitation (a, b) or PEG precipitation (c, d). Only the pellet-derived, heat-precipitated sample (b) shows a distinct peak near the column void volume at 7.2 mL (red box), consistent with an assembled T = 4 cage. The other three workflows elute between 13 and 25 mL, the range expected for monomeric and low-order species, and the two PEG routes recover little material (100 % ≈ 11 and 13 mAU, against 100 mAU in a). Together these traces indicate that assembled QtEnc-His partitions with the insoluble fraction and is discarded whenever the workflow proceeds from the clarified supernatant, which points to the His-tag compromising cage solubility. A₂₈₀ is normalised to the maximum of each trace after 1 mL (100 % = 100, 57, 11 and 13 mAU for a–d).

Fig S5. DLS of the crude HiPrep Sephacryl S-500 SEC isolation of wt QtEnc. a–e are different fractions of the SEC in Fig S9. Intensity and volume distributions are overlaid to contrast aggregation.

Fig S6. DLS of the crude HiPrep Sephacryl S-500 SEC isolation of QtEnc-TP-mScarlet. a–e are different fractions of the corresponding SEC (data not shown). Intensity and volume distributions are overlaid to contrast aggregation.

Fig S8. Analytical SEC of His-tagged QtEnc constructs after Ni-NTA purification. (a) QtEnc-His and (b) QtEnc-His-mScarlet, both expressed in E. coli BL21 (DE3) with 0.1 mM IPTG and filtered before injection onto a Superose 6 10/300 GL column. Neither trace shows a peak near the void volume (~8 mL); the signal starts at about 13 mL, so no assembled T = 4 cage was recovered by this workflow. A₂₈₀ is normalised to the maximum of each trace after 1 mL (100 % = 6.8 mAU in a, 8 mAU in b).

Fig S9. Crude SEC of three QtEnc variants under the revised purification protocol. Clarified lysate was subjected to heat precipitation followed by PEG precipitation, filtered, and applied directly to a HiPrep 16/60 Sephacryl S-500 HR column without prior Ni-NTA purification. (a) QtEnc, (b) QtEnc-TP and (c) QtEnc-mScarlet. All three traces begin to rise at about 40 mL and resolve into an early peak at 54–62 mL and a later peak at about 111 mL. The early peak elutes well ahead of the bulk of the soluble proteome and is the species assigned to assembled QtEnc. A₂₈₀ is normalised to the maximum of each trace after 1 mL (100 % = 180, 98 and 180 mAU for a–c).

Fig S10. Blue Native PAGE and the corresponding anti-His immunoblot across the QtEnc-His purification. (a) Coomassie-stained BN-PAGE and (b) anti-His immunoblot of the same steps. L, NativeMark unstained standard; 1, pellet after lysis centrifugation; 2, supernatant after lysis centrifugation; 3, pellet after heat precipitation; 4, supernatant after heat precipitation; 5, supernatant after filtration; 6, Amicon buffer-exchanged flow-through; 7, buffer-exchanged sample after concentration. Asterisks mark the fraction carried forward at each step (lanes 1, 4, 5, 7): the pellet recovered after lysis is the insoluble fraction taken onward, the route followed in a, while the paired heat-precipitation pellet (3) and the Amicon flow-through (6) were discarded; the lysis supernatant (2) was processed in a parallel branch not shown here. The dotted line marks the QtEnc-His species, which migrates above the 1236 kDa standard. In b the anti-His signal is confined to the same region at the top of the gel, with none detected in the resolved range.

Fig S11. SDS-PAGE across the QtEnc-His heat-precipitation workflow. Coomassie Brilliant Blue stained. L, prestained protein standard; − and +, whole cells before and after IPTG induction; 1, pellet after lysis centrifugation; 2, supernatant after lysis centrifugation; 3, pellet after heat precipitation; 4, supernatant after heat precipitation; 5, supernatant after filtration; 6, Amicon buffer-exchanged flow-through; 7, buffer-exchanged sample after concentration. Lanes 8–12 are the corresponding fractions of the parallel branch started from the lysis supernatant: 8, pellet after heat precipitation; 9, supernatant after heat precipitation; 10, supernatant after filtration; 11, Amicon flow-through; 12, sample after concentration. Asterisks mark the fraction carried forward at each step (lanes 1, 2, 4, 5, 7, 9, 10, 12): lysis splits into the pellet (1) and the supernatant (2), which were processed as parallel branches, while the paired heat-precipitation pellets (3, 8) and the Amicon flow-throughs (6, 11) were discarded. The dotted line marks the QtEnc-His monomer, which migrates just above the 35 kDa standard. Lane numbering matches that of the Blue Native PAGE. The image is a single contiguous crop of one scan; processing was limited to levelling, cropping, inversion and one linear intensity window applied to the whole image.

Fig S12. SDS-PAGE of Superose 6 fractions from the SEC run shown in Fig 4a. a covers fractions from 6.5 mL to 14.6 mL, b fractions from 15 mL to 19.5 mL. QtEnc-His monomers are present around 16–17 mL.

Fig S13. Stop-codon reversion assay plates showing induced (+IPTG), repressed (+glucose) and no-additive (basal) conditions. 50 µL of undiluted liquid culture was plated on 25 µg/mL chloramphenicol. Each condition shows growth. Triplicates are not shown.

Fig S14. SDS-PAGE of QtEnc-His Superose 6 fractions from the SEC run shown in Fig 4a. No QtEnc-His monomers can be identified around the elution-volume peak at ~8 mL.