Pfu DNA polymerase

Plasmid pDESTSIRV30, pDESTSIRV33 expressing the SIRV proteins (CAG38830 and CAG38833), pDESTAVRA expressing MRSA vraR protein (CAG40961) and pDESTFaBH2 expressing Pseudomonas aeruginosa FaBH2 protein (AAG06721)[28 (link)] were constructed using a modified Gateway technology with an N-terminal TEV protease cleavable His tag [29 (link)]. All the plasmids were propagated in DH5α E. coli cells (Stratagene, La Jolla) and plasmids were prepared using Qiagen miniprep kits (Qiagen, Germany). Pfu DNA polymerase, DpnI restriction enzyme are provided with QuikChange™ kit purchased from Stratagene, additional Pfu DNA polymerase was purchased from Promega when required. All the primers were synthesized by Eurogentec and simply purified by SePOP desalting. The melting temperature was calculated as T_m = 81.5 + 16.6(log([K+]/(1+0.7 [K+])) + 0.41(% [G+C]) – 500/(probe length in base) – 1.0(%mismatch) [30 (link)]. The T_{m pp} and T_{m no} were calculated for each primer. All primers and their T_{m no} and T_{m pp} are detailed in Table 1. PCR cycling was carried out using a Px2 thermal cycler (Thermo Electro Cooperation).
For single-site mutation, deletion or insertion, the PCR reaction of 50 μl contained 2–10 ng of template, 1 μM primer pair, 200 μM dNTPs and 3 units of Pfu DNA polymerase. The PCR cycles were initiated at 95°C for 5 minutes to denature the template DNA, followed by 12 amplification cycles. Each amplification cycle consisted of 95°C for 1 minute, T_{m no} -5°C for 1 minute and 72°C for 10 minutes or 15 minutes according to the length of the template constructs (about 500 bp per minute for Pfu DNA polymerase). The PCR cycles were finished with an annealing step at T_{m pp}-5 for 1 minute and an extension step at 72°C for 30 minutes. The PCR products were treated with 5 units of DpnI at 37°C for 2 hours and then 10 μl of each PCR reactions was analyzed by agarose gel electrophoresis. The full-length plasmid DNA was quantified by band density analysis against the 1636-bp band (equal to 10% of the mass applied to the gel) of the DNA ladders. An aliquot of 2 μl above PCR products, the PCR products generated using QuickChange™ or generated as described in [13 (link)] was transformed respectively into E. coli DH5α competent cells by heat shock. The transformed cells were spread on a Luria-Bertani (LB) plate containing antibiotics and incubated at 37°C over night. The number of colonies was counted and used as an indirect indication of PCR amplification efficiency. Four colonies from each plate were grown and the plasmid DNA was isolated. To verify the mutations, 500 ng of plasmid DNA was mixed with 50 pmole of T7 sequencing primer in a volume of 15 μl. DNA sequencing was carried out using the Sequencing Service, University of Dundee. For multiple site-directed mutations, deletions and insertions, the PCR was carried out in 50 μl of reaction containing 10 ng of template, 1 μM of each of the two primer pairs, 200 μM dNTPs and 3 units of Pfu DNA polymerase. The PCR cycles, DNA quantification, transformation and mutation verification were essentially the same as described above.

Optimized PCR enrichment conditions were performed by adding the following to 40 μl of eluted DNA from the adapter ligation reaction: 4 μl of Illumina F&R PE Enrichment Primers (Illumina, Inc., San Diego, CA, USA, catalogue number 1002290), 1 μl 100-mM dNTP mix (25 mM each; Agilent Technologies 200415), 6 μl 10× buffer (0.1 M KCl, 0.01 M MgSO₄.7H₂O, 0.01 M bovine serum albumin, 0.01 M (NH₄)₂SO₄, 0.2% Tris-HCl, 0.001% Triton X-100), 2 μl Pfu Ultra II Fusion HS DNA Polymerase (Agilent Technologies, catalogue number 600852) and 7 μl nuclease free water (VWR, Radnor, PA, USA, catalogue number PAP1193). Reactions were incubated on Eppendorf Mastercycler Pro thermalcyclers (Eppendorf, catalogue number 6321 000.515) for 120 s at 95°C, and cycled six times for 30 s at 95°C, 30 s at 65°C and 60 s at 72°C.

The association of purified Fis or Lrp proteins with the E. coli fim switch was measured using an electrophoretic mobility shift assay (EMSA). A 135 bp probe was amplified by PCR with Pfu polymerase (Stratagene), using the primer pair BSFORBIO and BSREVBIO (Table 2). The S. enterica spvR promoter was amplified as a 157 bp fragment using the primer pair, spvR11 and spvR14 (Table 2) [80 (link)], and this was used as a negative control for the Fis binding experiments [63 (link)]. The probes were then purified using a PCR clean-up kit (Roche Applied Science). The oligonucleotides had been ordered with 5′ biotinylated ends allowing for subsequent complex detection. Complexes were formed following incubation of amplified probe with increasing concentrations (0–270 nM) of purified His-tagged Fis [69 (link)] or 0–220 nM of purified His-tagged Lrp [38 (link)] for 15 min as described by the manufacturers of the Electrophoretic Mobility Shift Assay kit (Pierce). Competitive binding of purified Fis and Lrp was tested with Lrp being added in increasing concentrations to DNA that had been prebound with Fis at a constant concentration. Protein–DNA complexes were resolved by electrophoresis through a 7.5% polyacrylamide gel for 2 h at room temperature. The gel was then electrophoretically blotted and developed using the procedure recommended by the manufacturer (Pierce).

Sphingomonas sp. PAMC 26621 was kindly provided by the Polar and Alpine Microbial Collection of the Kore Polar Research Institute (Incheon, South Korea) [22 ]. The 333‐bp grxD (spgrx4) gene (NCBI ID: WP_010164075.1) was amplified from the genome of Sphingomonas sp. PAMC 26621 by PCR and subcloned into a TA vector (Enzynomics, Daejeon, South Korea). The TA–spgrx4 construct, digested by Nde I and BamH I, was subcloned into a pET28 vector (Novagen, Madison, WI, USA) and transformed into Escherichia coli BL21 (DE3). The pET28–spgrx4 construct was used as a template for site‐directed mutagenesis by PCR using pfu polymerase (PCR primers listed in Table S1). The PCR products were incubated with Dpn I at 37 °C for 1 h to remove the template plasmids before transformation into E. coli BL21 (DE3). The DNA sequence of the WT and mutant plasmids was confirmed by DNA sequencing.

Site-directed mutagenesis was performed with Pfu DNA polymerase using a protocol based on the QuickChange II site-directed mutagenesis kit (Agilent Technologies). The forward and reverse primers for each mutant are listed in Table S1.