Mycycler thermocycler

The c-MYC fragment previously PCR-amplified was used as a template for the LAMP reaction, performed as described by Veigas et al. [41 (link)]. This LAMP reaction requires four specific primers: a forward outer primer (FP), a backward outer primer (BP), a forward inner primer (FIP), and a backward inner primer (BIP) (see Table B1 for primers). LAMP primers for c-MYC were designed using Primer Explorer V4 (http://primerexplorer.jp/elamp4.0.0/). The reaction was carried out in a 10 µL reaction mixture containing 1.6 µM of FIP and BIP primers, 0.2 µM of B3 and F3 primers, 0.45 mM of dNTPs, 3 mM of MgCl₂, 0.8 M of Betaine, 700 µM of dNTPs, 1 X Bst Buffer, 1 X Evagreen dye, 1.8 U of Bst polymerase (New England Biolabs, Beverly, MA), and 1 µL (5–500,000 copies/µL) of template DNA; sterile water was used in place of DNA for the non-template control (NTC). The reaction was incubated at 65 °C for 60 min in a Bio-Rad MyCycler Thermocycler.

The sequence of C. perfringens ATCC 13124 from GenBank (accession number CP000246) was used to design primers for the amplification of genes involved in the metabolism of carbohydrates. The primers were designed to amplify the genes for trehalose utilization, treB, treC, and treR, the flanking sequences upstream and downstream of these genes and some regulatory genes (Table 1); DNA Star software was used to design primers. DNA was extracted according to a method described previously [35 (link)]. The 100 μl PCR mixture contained template DNA, 2 mM of dNTP mix, 0.5 μM of each forward and reverse primer, and 2.5 units of Taq polymerase from Roche or Applied Biosystems. The MyCycler thermocycler from Bio-Rad was used to amplify the genes. After initial denaturation at 95°C for 2 min, the genes were amplified during 30 cycles with the following parameters: 95°C for 15 sec, 52°C (or another appropriate temperature for the annealing of primers) for 30 sec, and 72°C for 90 sec, and final extension for 5 min at 72°C. The PCR products were purified from agarose gels and sequenced using an ABI Biosystems sequencer and dideoxy terminator.

Primer sets were designed to amplify MICB polymorphisms, which are present in exon 2, 3, 4, and 5 of the MICB gene. The forward primers were modified to have a phosphate group (P) at the 5′-end. The following primers were used in each single tube: sense 1, 5′-P-CAATGTGAAGTTATTTCCAGGAAGAAG-3′ from positions 7369 to 7395); antisense 1, 5′-CCAGGGTCGGTACCTGTTCT-3′ (from positions 8230 to 8249) for MICB exon 2 and 3; sense 2, 5′-P-CTGTTCCCTGCATCTCCCTTAGA-3′ (from positions 8765 to 8787); antisense 2, 5′-CCCATCTCCAGAAACTGTCCCC-3′ (from positions 9346 to 9368) for MICB exon 4 and 5 [14 (link)]. PCR was carried out in a reaction volume of 40μL containing 500 ng genomic DNA, 1× buffer (60 mmol/L Tris-Cl, 15 mmol/L ammonium sulfate, 100 mmol/L MgCl₂), 200 μmol/L dATP/dGTP/dCTP, 200 μmol/L dTTP/dUTP (dT:dU = 7:3), 4U Taq DNA polymerase (Bioprince; Enzynomics; Daejeon, Korea) and 10 μmol/L of each primer. The reactions were amplified using a Mycycler Thermo Cycler (Bio-rad Inc.; CA, USA) with the following conditions: initial denaturation of 98°C for 20 s; 8 cycles of denaturation at 98°C for 5 s, annealing at 65°C for 30 s, and extension at 72°C 30 s; followed by 37 cycles of denaturation at 98°C for 5 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s; and followed by a final extension at 72°C for 10 min. Then, the reactions were held at 20°C.

Site-directed mutagenesis of selected roseocin alpha precursor peptide residues was carried out using Agilent's QuikChange site-directed mutagenesis kit. Primers and methodology were designed using the procedure described by Liu and Naismith 2008 (link) study (Additional file 1: Figure S11). Desalted primers at a 0.05 µM scale were obtained from Sigma Genosys. The T_{m pp} (melting temperature of the primer-primer overlapping region) and T_{m no} (melting temperature of non-overlapping primer region) were calculated for each primer (Additional file 1: Table S7). PCR was carried out using Bio-Rad MyCycler thermocycler. The PCR products were treated with 5 units of DpnI at 37 °C for one hour, and the reaction was stopped by heating at 75 °C for 15 min. The PCR product was transformed into chemically competent E. coli DH5α. The mutations were confirmed through Sanger sequencing, and the plasmid was retransformed into E. coli BL21(DE3) for protein over-expression and in vivo post-translational modification by RosM (Singh et al. 2020 (link)), as per the details mentioned in Additional file 1.