FastDigest NcoI

Protocol tips

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	We digest the amplicon and pBAD-Ara::cmyc-His with FastDigest NcoI and FastDigest BgIII (both ThermoFischer Scientific) for 30min in Fast Digest buffer according to the supplier’s double digestion protocol. We treated the vector with alkaline phosphatase (NEB) at 37°C for 1h to avoid recircularization. A 1:5 vector:insert was incubated with T4 DNA Ligase (ThermoFischer Scientific) at 22°C O/N. The reaction product was dialysed to MiliQ water for 1h using dialysis disks.

Protocol tips

We digest the amplicon and pBAD-Ara::cmyc-His with FastDigest NcoI and FastDigest BgIII (both ThermoFischer Scientific) for 30min in Fast Digest buffer according to the supplier’s double digestion protocol. We treated the vector with alkaline phosphatase (NEB) at 37°C for 1h to avoid recircularization. A 1:5 vector:insert was incubated with T4 DNA Ligase (ThermoFischer Scientific) at 22°C O/N. The reaction product was dialysed to MiliQ water for 1h using dialysis disks.

Publication protocol

"The nucleotide sequences for BtsCI restriction enzyme (btsCIR) and the respective methyltransferase (btsCIM) were obtained from NCBI (Accession no GQ449683) and the plasmids were obtained from New England Biolabs (NEB). We decided to reclone btsciR into pBAD-Ara::cmyc-His (ampicilinR). We started by preparing the insert by PCR amplification of the btsciR sequence with the following oligos:

EF_NcoI_BtsCIR_Fw: 5´CCACCATGGGTAAACGAATTTTATACTTGCTAACT3´

EF_BglII_ BtsCIR_NO STOP_RV: 5´CCAAGATCTCAGAAAAACAGCGCTTCCAT3´ and a PCR program starting with 30s at 98°C followed by 30 cycles of 10s at 98°C, 30s at 68°C and 72°C for 45s, and finishing with 10min at 72°C. We used 0,2μL (0.4U) of Phusion High-Fidelity DNA polymerase (ThermoFischer Scientific) in 1μL of DNA template with 1μL of each of the aforementioned primers (final concentration of 0.5μM), 4μL of HF Buffer, 0.4μL of 10mM dNTPs and water up 20μL. It is important to notice that in the EF_BglII_ BtsCIR_NO STOP_RV oligo we do not include the STOP codon of the gene in order to obtain a fusion with a c-myc fragment and a His-tag, which were used for purification later. We digest the amplicon and pBAD-Ara::cmyc-His with FastDigest NcoI and FastDigest BgIII (both ThermoFischer Scientific) for 30min in Fast Digest buffer according to the supplier’s double digestion protocol. We treated the vector with alkaline phosphatase (NEB) at 37°C for 1h to avoid recircularization. A 1:5 vector:insert was incubated with T4 DNA Ligase (ThermoFischer Scientific) at 22°C O/N. The reaction product was dialysed to MiliQ water for 1h using dialysis disks.

The methyltransferase gene was obtained in the vector pACYC184. We electroporated pACYC184:btsciM (chloramphenicolR) into Escherichia coli D1210 (F- mcrB mrr hsdS20(rB- mB-) recA13 leuB6 ara-14 proA2 lacY1 galK2 xyl-5 mtl-1 rpsL20(SmR) glnV44 λ- lacIq lacY+) and allowed the bacteria to grow for 1h in LB before plating in agar with chloramphenicol (25μg/mL). After confirmation of a successful transformation by Sanger sequencing, we electroporated the resulting bacteria (D1210+pACYC184::btsciM) with the pBAD-Ara::btsciR-cmyc-His-tag construct and repeated the growth procedure by plating in agar with chloramphenicol (25μg/mL) and ampicillin (100 μg/mL). Glucose was also added to the plates (0.2% final concentration) to avoid leakage from the pBAD-Ara promoter."

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