Results#
Read this first
The continuous evolution campaign has not been run. What follows is the state of the build: what is verified, what is still failing, and what is being done about it. We are reporting the negative results because they are the honest description of where a season's work actually got to, and because the troubleshooting is the part with transferable content.
Where things stand#
| Subsystem | Status |
|---|---|
| Shell expression (His-QtEnc) | Working — confirmed by SDS-PAGE and anti-His immunoblot |
| Shell assembly into 42 nm cages | Not yet demonstrated — see below |
| Selection plasmid, guide truncation series | Built and sequence-verified (non-targeting, 10, 11, 14, 17, 20 nt) |
| dCas9 cloned out of source plasmid | Done |
| Mutation plasmid series | In assembly |
| Selection plasmid screen | Not started |
| Continuous evolution | Not started |
Expression works; assembly has not been shown#
QtEncapsulin constructs express. A band of the expected monomer mass appears after IPTG induction and is confirmed by anti-His immunoblotting.
Assembled shells are a different matter. Across repeated purifications, size exclusion chromatography on Superose 6 gave only a shallow feature near the void volume, with the bulk of the material eluting at 16–20 mL — consistent with monomers and small oligomers, not with a 7.7 MDa cage. Dynamic light scattering of the pooled fractions never yielded a population consistent with the expected 42 nm diameter; the samples read as polydisperse and dilute.
The negative result, stated plainly
Between May and September we did not obtain evidence of assembled QtEncapsulin. Every SEC run and every DLS measurement is consistent with the protein being expressed and not assembling, or assembling and not surviving the purification.
What we changed in response#
Four hypotheses were separated and tested one at a time.
Initial inductions used 1 mM IPTG. Published protocols for this shell use 0.1 mM, and over-strong induction is a standard cause of inclusion-body formation. Induction was dropped to 0.1 mM, expression temperature to 18 °C, and 1 % glucose was added to the growth medium to suppress leaky expression before induction.
The hexahistidine tag is joined to the shell protein through a GS linker. If the tag is occluded in the assembled cage, Ni-NTA would preferentially recover unassembled monomer and discard exactly the species we are looking for — which would explain a monomer-rich elution profile from a culture that is assembling normally. Constructs with a direct terminal His tag, which the supplementary material of published work reports as assembling successfully, were built to test this.
Sonication was producing foaming, and the clarification spin may have been pelleting intact cages along with debris. Spin speeds were varied and aliquots retained at every step so that the losses could be located on a gel rather than guessed at.
Two routes were run on split aliquots of the same lysate: PEG-8000/NaCl precipitation, and heat precipitation exploiting the shell's thermostability. Aliquot-by-aliquot A280 and SDS-PAGE showed most material partitioning into the pellet after lysis, which is itself informative.
Selection plasmid screen#
The guide truncation series is built and sequence-confirmed. The screen that identifies which guide places unrescued cells just below the kanamycin survival threshold has not yet been run.
Kanamycin plates have been prepared at 50 and 200 µg/mL, and with nitrotetrazolium blue as a colorimetric viability readout, in anticipation of it.
Sequenced variants#
None — the campaign has not run, so there are no evolved variants to report.
What would change the picture#
A single clean SEC trace with a peak at the void volume, corroborated by DLS and negative-stain EM, unblocks everything downstream. The selection circuit does not depend on the purification working — the shell only has to assemble in vivo for the selection to function — but without an assembly assay we cannot tell an evolved improvement from noise, and we cannot validate that the OR gate is capturing what we think it is.