Evolution cycles#

MutaT7 induction#

Both plasmids are co-transformed into the MutaT7 host strain and maintained under dual selection. Inducing the T7 RNA polymerase–deaminase fusion starts hypermutation of the T7-flanked encapsulin cassette, and of nothing else.

Two practical notes carried over from the build. The MutaT7 strain transforms poorly compared with DH10β, so competent-cell aliquots are made at 200 µL rather than 100 µL per transformation. And glucose is included in growth medium before induction to suppress leaky expression.

Selection stringency#

One set-point, one ramp.

Set-point — guide length. The truncation series spans a range of passage probabilities. The working guide is chosen in a plate screen as the one whose unrescued residual resistance sits just below the survival threshold, so a modest gain in capture decides whether a cell grows.

Ramp — kanamycin. The dose is raised between passages, starting from 50 µg/mL, each step demanding a larger sequestered fraction. Where finer adjustment is wanted the guide is exchanged for a longer one instead of, or alongside, raising the dose.

Nitrotetrazolium blue plates are used as a colorimetric viability readout alongside the kanamycin series.

Passaging schedule#

Continuous culture in a turbidostat (Pioreactor), so that selection acts on growth rate every generation rather than in discrete plate-based rounds. At each passage:

1. Sample and archive. Glycerol stock, so any passage can be returned to.
2. Sequence the mutagenised cassette. Follows which mutations are enriching, and at what point.
3. Re-sequence the selection plasmid. Catches escape — loss-of-function in dCas9, the sgRNA or their promoters — which would restore resistance without improving encapsulation.
4. Decide the next stringency step from the growth rate at the current dose.

What counts as a result#

An enriched allele is not a result until it is recloned into the expression vector and characterised independently: assembly by SEC and DLS, capture by the co-encapsulation assays, and specificity by the single-handle controls. The selection identifies candidates. It does not, on its own, demonstrate that anything was encapsulated.