This week, our project began moving from computational design into practical experimental planning. Our central goal remains to evolve intrinsically disordered protein (IDP) tags that can help other proteins remain soluble and functional. To test this idea, however, we first needed to turn our proposed libraries and selection systems into workflows that were realistic within our time, budget and available equipment.
One important decision concerned how to introduce diversity. We compared commercial error-prone PCR kits with a custom method based on standard Taq polymerase and altered manganese and magnesium concentrations. Commercial kits offered convenience, but their cost would have limited the number of experiments we could run. We therefore developed a lower-cost error-prone PCR strategy that can be optimised experimentally. This also led us to think more carefully about the balance between mutation rate and usable DNA yield: more mutations are not necessarily helpful if amplification becomes unreliable.
We also revised how our designed libraries would be ordered. Double-stranded degenerate DNA was prohibitively expensive, so we moved towards single-stranded synthesis followed by conversion to double-stranded DNA in the laboratory. This reduced costs while keeping the broader design-build-test-learn cycle intact, although it introduced an additional step that would later need careful validation.
At the same time, the team refined both experimental branches. For the beta-lactamase system, we mapped the stages required for plasmid construction, transformation and selection, and began questioning whether liquid selection would give a reliable measure of improved protein performance. For the tardigrade-inspired system, we narrowed the possible stress assays and developed a practical desiccation setup using filter paper and a desiccator. We also began considering controls that could distinguish genuine protection from simple recovery under favourable conditions.
The week ended with the first preparations for experimental work: antibiotic plates were poured, bacterial strains were cultured and chemically competent cells were produced. We also revised our schedule and accepted that two well-supported rounds of evolution would be more realistic than three rushed rounds.
The main outcome of the week was not a single experiment, but a more grounded project. Our early ideas had become a set of testable workflows, with clearer constraints and more explicit assumptions to examine in the weeks ahead.