This serves as a chronological record of your team's progress throughout the season. It documents your daily activities, experiments, discussions, and decisions.
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.
Date-stamped entries, including the bad days — on the argument that iGEM judges care about reproducibility, and this is what it looks like when a team actually means it.
PCR-amplified PETase ORF from a synthesised gBlock (832 bp). First attempt: a primer-dimer band swamped the signal. Lowering the annealing temperature made it worse. Re-ordering primers with a 3' clamp (G/C lock) worked first time at 65°C. Notebook lesson: when in doubt, the primers are wrong, not the polymerase. Result: a single clean band at ~830 bp, sequence-verified on all 3 colonies, stocked in glycerol at −80°C.
Attempted to induce Ag43-PETase with 0.2% arabinose for 4 hours, expecting mass loss but not growth arrest. OD600 at induction was 0.62; after 90 minutes the cultures stopped growing, and by 4 hours two of three flasks were visibly cloudy with lysate. Acridine-orange staining confirmed compromised outer membrane in over 40% of cells. "It looked fine at t=0. It looked terrible at t=2h. We did not see this coming." — lab note. Decision: change anchor system; Lpp-OmpA and INPNC moved to the head of the queue.
Dropped Ag43. Built two fusions in parallel — Lpp-OmpA-PETase and INPNC-PETase — on the reasoning that it wasn't yet known which would be membrane-safe at activity-relevant levels, so running both in parallel produces two real data points instead of guessing.
Plated 10⁷ CFU of each switch-bearing strain onto selective media at the OFF condition. Day-1 result: the temperature switch showed a ~10⁻⁴ escape rate (acceptable for an unoptimised single switch); the light switch showed ~10⁻³, worse than predicted, hypothesised to be photoreceptor saturation. Joint estimate so far: 10⁻⁵ — contingent on the independence claim holding up.
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