This week reminded us that a successful-looking experiment is only useful when the identity of the material is certain. Much of our work therefore focused on verifying plasmids, improving error-prone PCR and strengthening the controls around our assays.
The beta-lactamase construct had produced transformed colonies, allowing us to extract plasmids and prepare for a second linearisation. Adjusting the primer concentrations improved this PCR and produced clear bands. Sequencing confirmed that beta-lactamase had been inserted correctly into one construct, supporting the next assembly stage. However, another sequencing result showed that a sample believed to contain the original pBAD plasmid was actually the beta-lactamase-containing version. A growth-curve dry run also behaved more like DH10B than the intended BL21 strain.
Together, these results shifted our emphasis from simply progressing through the protocol to verifying strain and plasmid identity at each stage. Fluorescence under arabinose induction, selective plating, diagnostic PCR and sequencing became complementary checks rather than optional confirmation at the end.
The error-prone PCR branch faced a different problem. We tested DMSO, betaine and extension time to reduce mispriming and identified conditions that initially produced the expected fragment. Yet the next round again generated smears or failed amplification, even when repeated by another team member. This suggested that the problem was inherent to the reaction or template rather than a single pipetting mistake. We therefore began developing a computational tool to analyse possible mispriming sites and paused plans to model mutation bias until enough later-round sequencing data could be generated.
We also continued refining the tardigrade-inspired experiments. Recovery and plating conditions were varied after the first desiccation dry run produced either no colonies or lawns that were too dense to interpret. The team learned to use a plate reader and drafted a growth-curve protocol, but contamination and a power interruption made the first run inconclusive.
This week did not produce a simple forward march. Instead, it exposed several hidden assumptions about what was in our tubes and which organisms were being measured. Finding these problems changed how we approached the project: identity checks, controls and reproducibility now needed to be built into the workflow before selection results could be interpreted with confidence.