This week, unexpected colonies in control plates forced us to reassess the reliability of our assembly workflow. At the same time, we made progress towards sequencing our first constructed library and clarified how the strongest variants could eventually be validated beyond the initial selection system.
The results from an earlier Gibson assembly were difficult to interpret. The positive control produced no colonies on plates, the water control produced many small colonies and growth appeared in several liquid cultures. Because the controls did not behave as expected, we could not confidently attribute growth to successful assembly of the SSB library.
Rather than continuing to troubleshoot on uncertain foundations, the team introduced stricter contamination checks. Media flasks would be labelled and incubated after use, work surfaces and pipettes would be sterilised daily, and suspect consumables were replaced. We also repeated the Gibson assembly with support from a neighbouring laboratory. This allowed the project to move forward while preserving time for the selection stage.
Improved purification protocols produced DNA with more consistent purity, and the SSB library and original SSB control were assembled and transformed. Plasmids were extracted at usable concentrations, and the library sample was sent for sequencing. Sending the constructed library for sequencing was a key checkpoint: before applying selection, we need to know whether the sequences we designed are actually represented in the biological population.
The team also prepared degenerate De Novo and NEXT tags and calculated the DNA ratios required for further assemblies. Low yields from one gel-extraction kit reinforced an earlier lesson, and we chose an alternative purification method rather than allowing the same bottleneck to dominate another week.
Alongside construction, we refined our plans for final validation. Several possible target proteins were considered for testing whether selected IDP tags improve both solubility and function. After reviewing experimental precedent and feasibility, citrate synthase and lactate dehydrogenase emerged as practical candidates for the tardigrade-inspired assays. This complements the high-throughput beta-lactamase selection by providing more direct measurements of enzyme protection or activity retention.
The week brought the project closer to selection, but its most important outcome was stronger experimental discipline. Reliable controls, contamination monitoring, sequence verification and meaningful validation targets are what allow an apparent improvement to become a defensible result.