Phusion high fidelity or taq dna polymerase

Chromosomal DNA was extracted from B. thuringiensis using the Puregene Yeast/Bact. Kit B (QIAgen, France). Plasmid DNA was extracted from E. coli by standard alkaline lysis by using QIAprep spin columns (QIAgen, France). Restriction enzymes and T4 DNA ligase were used as recommended by the manufacturer (New England Biolabs). Oligonucleotide primers (Supplementary Table 1) were synthesized by Sigma Proligo (Paris, France). PCRs were performed in an Applied Biosystems 2720 Thermal cycler (Applied Biosystem, United States) with Phusion High-Fidelity or Taq DNA Polymerase (New England Biolabs). Amplified DNA fragments were purified with the QIAquick PCR purification kit (QIAgen, France). Digested DNA fragments were separated by electrophoresis and purified from agarose gels using the QIAquick gel extraction kit (QIAgen, France). All constructions were confirmed by DNA sequencing by GATC Biotech (Konstanz, Deutschland).

Chromosomal DNA was extracted from B. cereus cells with the Puregene Yeast/Bact. Kit B (QIAgen). Plasmid DNA was extracted from E. coli and B. cereus using QIAprep spin columns (QIAgen). For B. cereus, 5 mg ml⁻¹ of lysozyme was added and cells were incubated at 37°C for 1 h. Restriction enzymes and T4 DNA ligase were used according to the manufacturer's instructions (New England Biolabs). Oligonucleotide primers (Table 4) were synthesized by Sigma-Proligo. PCRs were performed in an Applied Biosystem 2720 Thermak cycler (Applied Biosystem) with Phusion High-Fidelity or Taq DNA Polymerase (New England Biolabs). Amplified fragments were purified using the QIAquick PCR purification Kit (QIAgen). Digested DNA fragments were separated by electrophoresis on 0.8% agarose gels and extracted from gels using the QIAquick gel extraction Kit (QIAgen). Nucleotide sequences were determined by Beckman Coulter Genomics.
The thermosensitive plasmids pMAD [78] (link) and pRN5101 [79] (link) were used for homologous recombination. The low-copy-number plasmid pHT304 [80] (link) was used for complementation experiments with wild-type entA gene under its own promoter. The vector pGEX6P1 (GE Healthcare) was used to overproduce Glutathione S-transferase (GST)-tagged protein under the control of a tac promoter. All the plasmids used in this study are reported in Table 3.