With the identities of our main plasmids and strains rechecked, this week we returned to the central engineering challenge: converting designed IDP sequences into a physical library that could be inserted and tested.
Selective and fluorescent plating confirmed that the required transformed cells had been obtained. We then attempted to convert the single-stranded degenerate tag oligos into double-stranded DNA and used the SSB library in a Gibson assembly. Although the assembly mixture appeared to contain abundant DNA, the transformed cells initially failed to grow. Gel analysis later showed that the direct single-strand-to-double-strand conversion had produced a smear, whereas an amplified product gave a clear band.
This result changed the immediate workflow. Rather than relying on direct conversion alone, we moved to amplification before Gibson assembly, then repeated the assembly and transformation with positive and negative controls. The failure was therefore informative: it connected a downstream transformation result to an earlier library-preparation step and showed why concentration measurements alone cannot confirm that a DNA sample contains the intended product.
The error-prone PCR branch also continued, although the project timeline forced us to narrow its scope. We established that the reaction could be reproduced and compared purified with unpurified templates. Purified material performed better, while unpurified reactions produced a short unwanted product. Further mutation rounds were completed for SSB and NEXT, but limited time meant that the number of rounds and variants entering selection would need to be prioritised.
For the tardigrade-inspired branch, we finalised protocols for heat shock and osmotic stress and adjusted the desiccation setup after thin filter paper curled during drying. These practical changes were guided by the same principle as our library work: the assay must be physically reproducible before it can tell us whether an IDP provides protection.
The team also met with potential reagent partners, securing support that could reduce the cost of future orders and troubleshooting.
By the end of the week, we had not yet completed selection, but we had crossed an important boundary. The designed sequence libraries were no longer only computational objects; they were being amplified, assembled and transformed. Each failure was helping us define the conditions needed to preserve the link between sequence design and biological testing.