Question
PET bottles take centuries to break down. Enzymes that cut PET into its monomers could make recycling work at low temperature. We want to know whether an engineered PETase is good enough to depolymerise untreated bottle film within a week, in our hands, with our plate reader and HPLC.
Sources
Yoshida et al. (2016) A bacterium that degrades and assimilates poly(ethylene terephthalate). Science 351, 1196–1199The bacterium Ideonella sakaiensis and the two enzymes that convert PET into its monomers.doi.org/10.1126/science.aad6359
Lu et al. (2022) Machine learning-aided engineering of hydrolases for PET depolymerization. Nature 604, 662–667An engineered PETase variant reported to depolymerise post-consumer PET at moderate temperature.doi.org/10.1038/s41586-022-04599-z
From a planning callThe voice colleague pointed out that both papers measure products by HPLC, while our draft only weighed the film. We added HPLC sampling.
Hypothesis
Hypothesis · A. KayaThe engineered PETase releases at least three times more product from bottle film at 50 °C than the wild type in 7 days.Because Lu et al. report much higher activity on untreated post-consumer PET. Outcome ≥ 3× products by HPLC at day 7, 3 replicates.
Protocol
PETase expression and film assay · v2 · generated from the two papers, edited by J. Meyer
- Clone codon-optimised wild-type and engineered genes into pET-28a (N-terminal His-tag); screen colonies by colony PCR, insert ≈ 0.9 kb
- Express in BL21(DE3): grow to OD600 0.6, induce with 0.1 mM IPTG, 18 °C, 16 h
- Purify on Ni-NTA; check purity on SDS-PAGE; measure concentration by A280
- Punch 6 mm discs from one bottle; wash; weigh each disc
- Incubate discs with enzyme in buffer at 40 °C or 50 °C; 3 discs per condition per run
- Sample on days 1, 2, 4 and 7; quench; measure released products by HPLC
Decided before run 1Temperatures: 40 °C and 50 °C (not 30 °C; plate shaker only goes to 50 °C).
Runs
| Run | By | Date | Deviations |
|---|---|---|---|
| 1 · cloning check | JM | 4 Sep | – |
| 2 · expression + assay | AK | 10–17 Sep | Induced at OD600 0.72, planned 0.6 · disc mass varied ±12%, results normalised per mg |
| 3 · repeat assay | LS | 17–24 Sep | – |
Results
Observation · day 4 · L. SchmidtDiscs with the engineered enzyme at 50 °C turned opaque and brittle; wild-type discs looked unchanged.
ResultAt day 7, the engineered enzyme at 50 °C released 7.9 mM products versus 1.9 mM for the wild type at 50 °C: 4.1×. The wild type did slightly better at 40 °C than at 50 °C.
Conclusion
The hypothesis holds in this example: more than three times the product in a week, on untreated bottle film. Next we test higher-crystallinity film from a second bottle brand, and whether the enzyme survives a second week.
Review AI · GPT (OpenAI)The comparison is fair: same discs, same buffer, same days. Disc mass varied in run 2; results are normalised to 10 mg of film, as the figure states. Run 3 alone would support the same conclusion.Drafted by GPT (OpenAI) from this notebook's runs and both papers · checked and added by J. Meyer
Provenance
SignedEvery block carries its author's account signature.
TimestampedRecords are anchored with an independent timestamp.
AI contentTwo blocks are AI-written; each names the model and the person who added it.
SharingSealed page: the key is in the link, the server stores only ciphertext.
page PET-01 · 14 blocks · 3 authors · exported 25 Sep 2026