DNA Methylation Cycle for Bacteria Study

What I need done

I need a small, well-documented wet-lab experiment run and reported. It is straightforward benchwork using commercially available kits — but as far as we can establish it has never been published, so careful documentation matters more than speed.

The experiment. Take E. coli genomic DNA, add methyl marks at CpG sites with an enzyme, remove them again, then re-add them. Repeat for up to 10 cycles. At each cycle, measure two things:

1. How completely the marks were removed (% residual methylation after each erase)
2. Whether the DNA is still intact (fragment size / degradation across cycles)

Protocol outline

- Starting material: E. coli K-12 genomic DNA. Preferably a dam⁻/dcm⁻ strain (e.g. JM110 / GM2929), which starts with no CpG methylation. Confirm the zero baseline before cycle 1.
- Write: CpG methyltransferase M.SssI (NEB M0226) + SAM, per supplier protocol.
- Measure methylation. Either method is acceptable — tell me which you'd use and why:
- Option A (lower cost): methylation-sensitive restriction digest — HpaII (blocked by CpG methylation) vs MspI (not blocked) — with gel or TapeStation readout
- Option B (higher resolution): nanopore sequencing, EM-seq, or bisulfite sequencing
- Erase: I'm open on chemistry and want your input. Candidate routes: TET2 oxidation (NEB M1524) followed by base-excision repair enzymes (TDG, APE1, POLβ, ligase); or an alternative you consider more likely to work. Note that TET2 alone stops at 5-carboxycytosine and does not return the base to unmodified cytosine — the erase must go all the way back.
- Re-write with M.SssI and repeat.
- Integrity check at every cycle: TapeStation, Bioanalyzer, or pulsed-field gel. Report fragment size distribution, not just "intact/degraded".
- Run a buffer-only control held at 37 °C for the same duration, so incubation damage is separated from erase-chemistry damage.

Checkpoints: report after cycles 1, 2, 5 and 10.

Deliverables

1. Written protocol exactly as performed, including every deviation
2. Raw data files and instrument outputs
3. Gel / TapeStation images for every timepoint
4. A results table: cycle number → % methylation after write → % residual after erase → DNA fragment size
5. A short plain-language summary of what worked and what didn't
6. Photographs of key steps

Requirements

- Access to a molecular biology lab (university, CRO, or private)
- Experience with enzymatic DNA modification; DNA methylation experience strongly preferred
- Able to source reagents from NEB or equivalent, or advise me so I can order them
- Willing to report negative results honestly — a clear "it stops working at cycle 3" is a valuable outcome, not a failure

Important

This is exploratory. Nobody has published this cycling experiment, so I am not expecting it to work perfectly. I want accurate measurement, not a good-looking result. If erase efficiency is poor, that is exactly what I need to know, and it will be reported as such.

Please include in your proposal

1. What lab you have access to, and what equipment
2. Which methylation-measurement method you'd choose, and why
3. Which erase chemistry you'd try first
4. Estimated reagent cost (billed separately or included?)
5. Realistic timeline for cycles 1–2, and for the full 10
6. Whether you've worked with methyltransferases or TET enzymes before