Describe the research, experiments, and protocols you used in your project. It is designed to provide sufficient information for other teams to replicate our work.
Provide comprehensive, step-by-step protocols for all experiments conducted. Include a list of materials, reagents, and equipment used. Specify concentrations, volumes, incubation times, temperatures, and other critical parameters. Document any modifications or optimizations made to standard protocols.
Explain the purpose of each experiment and its relevance to your project goals. Describe the experimental design, including controls and replicates. Provide a clear rationale for the chosen methods and approaches.
Document any troubleshooting steps taken and optimizations made during the experimental process. Share any lessons learned that could benefit future teams.
The section below is a separate, independently-written draft, produced by a team member outside the main documentation process. Kept here for reference and comparison; not yet fact-checked by team leadership.
Twelve protocols across four families, on the principle that reproducibility is a forcing function — if a protocol doesn't appear here, it didn't happen. A protocol is only added to this list once it has been run by two team members on different days and produced reproducible results; drafts live elsewhere until they meet that bar.
Gene assembly, primer design, sequence verification. Gibson assembly variants for PETase, MHETase, and the anchor fusions. Primer design rules: 3' clamp, Tm matching within ±2°C, no homopolymer runs of 4 or more.
Shake-flask induction, membrane fractionation, activity assays. Standard pBAD and pTrc induction at 30°C and 37°C. Spheroplast preparation for outer-membrane localisation tests. pNPB hydrolysis used as an activity surrogate.
PET-film disc preparation, mass-loss quantitation, HPLC monomer detection. 100 mg PET film discs from commercial drink-bottle stock, surface-cleaned with ethanol, pre-weighed. 14-day incubation at 25°C. Mass loss plus reverse-phase HPLC for MHET, TPA, and EG quantitation.
Kill-switch escape-rate counting and membrane-integrity staining. Plate-based escape-rate counting at 24h, 48h, and 72h. Acridine-orange membrane stain with brightfield overlay for live/dead discrimination.
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