Determination of Adenine Methylation at GATC Sites in lambda DNA.

Dam protein was overexpressed, in E. coli cells expressing TetR constitutively (MG1655 Z1X strain, overexpressing tetR, lacI and araC), by addition of the plasmid pLTetO1-dam and growing the cells on LB + 10 mM MgSO4 + 0.2% Maltose + a gradient of concentration of anhydrotetracycline (aTc). TetO/tetR system allows a strong repression of the locus controlled by TetO, allowed by TetR; aTc is a strong competitor for TetR binding, allowing expression of the locus.

lambda phage was multiplied on liquid cultures of MD301 strain with varying levels of aTc concentration (0,5 or 50 µM), starting from a hundred phages (to be sure not to have a loss of function cII mutant in the inoculum) and 4 x105 bacteria. Phage and bacteria were initially mixed in 10 µL of LB supplemented with MgSO4 10 mM and maltose at 0.2% and incubated for one hour at 37°C. After one hour, 1 ml of LB at 37°C was added, and the phage/ bacteria mixture was incubated for another two hours at 37°C with agitation, after what it was diluted in 9 ml of LB and incubated with agitation until lysis occurred (monitored by a drop of optical density). Culture was centrifuged at 4500 g for 10 minutes and the supernatant filtered with a 0.2 µm size pore filter, and kept at 4°C until use.

Phage DNA was extracted from 5 ml of lysate. The lysates were centrifugated 20 min at 4500g, filtered with a 0.2 μm filter, treated with Turbo DNase™ (Invitrogen™, US) and RNase I to remove the host contaminant DNA and RNA and precipitated with polyethylene glycol 10% and NaCl 0.5 M for 6 hours. The lysates were centrifuged and the pellets were resuspended in SM buffer, treated with proteinase K, and the phage DNA was purified using a standard phenol:chloroform procedure. DNA purity and concentration were checked with the NanoDrop™ 2000 spectrophotometer (Thermo Scientific™, US) and the Qubit4™ fluorometer (Invitrogen™, US), respectively. DNA integrity was checked by 1% agarose gel electrophoresis. DNA was sent for sequencing at the I2BC sequencing facility. DNA was sequenced with a GridION X5 (Oxford Nanopore Technologies, UK) and long-reads were analysed with modkit v0.3.1 (https://github.com/nanoporetech/modkit) using per default parameters. The results of the comparative mode were used, using DNA from a dam knockout condition (MVEC232) as reference (methylation level fixed at 0). An additional control was added, lambda phage grown on WT MG1655 were prepared by induction of lambda at 42°C for 10 min, followed by 1h incubation at 37°C.

As a positive control, we used lysogenic cells for lambda, deleted for dam, harbouring pLTetO1-dam and Tn10-tetR-tetA (lambda plTet-dam). This genotype results in an overexpression of dam independently of the aTc concentration.

Modkit, 0.3.1

fastqc, 0.12.1