At this stage, our main focus was turning a broad project idea into something we could actually order, clone, and test.
We began by narrowing our choice of target protein. An earlier chloramphenicol-related system became less attractive because of structural complications, so we shifted our attention toward an ampicillin-resistance enzyme as a more practical selectable target. The idea was to use a weakened or aggregation-prone version of the enzyme, then ask whether different IDP tags could improve its performance.
We also started refining our tag-generation strategy. Rather than mutating entire tags blindly, we planned to identify likely mutation regions first, then feed selected variants into our reverse-translation workflow. This was important because very large degenerate libraries quickly become impossible to sample properly.
A major discussion point was the trade-off between coverage and off-target sequences. Degenerate codons can represent many variants, but they can also generate sequences we did not intend to test. Our early goal was therefore to reduce unnecessary variation while still keeping enough diversity for selection.
By the end of this stage, we had early DNA designs for SSB, NEXT/N11, and the beta-lactamase target, but several decisions still needed confirmation: whether the target system was final, whether the mutation strategy was acceptable, and whether our reverse-translation approach gave a sensible balance between library size and sequence coverage.