This week, we refined the experimental direction for our IDP solubility-tag project. The beta-lactamase branch is moving closer to wet-lab work, with sequences finalised and ordering planned. Reagents for the next stage of cloning have also been arranged.
A major decision was to simplify the tardigrade branch. Instead of using LDH as the main selection system, we are moving towards survival-based testing after desiccation or freeze-thaw stress. This should make the workflow more consistent with the rest of the project and easier to scale.
We also updated the tardigrade tag design. The team plans to use a species where truncated tardigrade proteins have already been reported to retain function, and to focus first-round mutations on a better-defined motif. The current design rationale is based on helical propensity, controlled hydrophobicity, and avoiding changes that could harm bacterial growth.
Finally, we discussed the difference between selection and validation. Selection can tell us which variants survive or become enriched, but validation is needed to show why. Our next challenge is to connect survival improvements back to protein solubility using assays such as soluble-versus-insoluble beta-lactamase measurements.
Overall, the project is moving from broad design planning into more concrete ordering, cloning, selection, and validation decisions.