Xhoi

Primer sets for PCR amplification were designed with Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi)according to the target sequences (Supplementary Table 1) and synthesized by Sigma-Aldrich. One microgram of total RNA was reverse-transcribed for 50 min at 42°C with SuperScriptTM Reverse Transcriptase (Life Technologies) and the reverse primer. One or two μL were then PCR amplified in a 50 μL reaction mixture containing 200 nM primers, 200 μM dNTPs, 1.25 U AmpliTaq Gold and 1.5mM MgCl_2.The thermal condition was: denaturation at 94°C for 10 min, amplification for 35 cycles (denaturation at 95°C for 30s, annealing at 55°C for 30s, extension at 72°C for 30s), and final extension at 72°C for 10 min. The PCR products were separated by gel electrophoresis in a 1.2% agarose gel.
PCR products were gel-purified with Ultrafree DA(Millipore), concentrated with Microcon YM-50 Device (Millipore), and digested for 2h at 37°C with 10U of either PstI, XhoI, PvuII or SacI (Roche Applied Science). The digested fragments were separated by gel electrophoresis in 1.2% agarose gel.

Recombinant plasmids from the metagenomic libraries constructed in E. coli DH10B cells were extracted using the QIAprep Spin Miniprep kit (Qiagen) and 100 ng of them were used to transform electrocompetent cells of E. coli DH10B ΔcsdA and DH10B ΔcsdA Δrnr, which had been previously prepared according to Dower et al., 1988 (link). To amplify the libraries, after electroporation, the transformed cells were grown in liquid LB-Ap medium at 37°C to increase the number of viable cells around 10⁴ times. To select for cold-tolerant clones, a total of 3 × 10⁶ recombinant clones from each library were spread on LB agar supplemented with Ap, X-Gal, and IPTG (around 10⁵ cells per plate) and incubated at 15°C during 3–5 days until the appearance of colonies. To ensure that the resistance phenotype was not due to the presence of spontaneous chromosomal mutations, resistant colonies were pooled, and their plasmid DNA were isolated and used to transform DH10B ΔcsdA or DH10B ΔcsdA Δrnr cells. The new retransformed clones were grown again at 15°C on LB agar (Ap, X-Gal, IPTG) medium. Cold-tolerant clones were selected and their isolated recombinant plasmids were digested with XhoI and XbaI (Roche) to identify those which are unique in their restriction patterns.

A 1311-bp LIC13300 DNA fragment was amplified using oligomers LIC13300-F 5′-GGAATTCCATATGAGAGAAATCAAAACAGTAACAG-3′ and LIC13300-R 5′-CCGCTCGAGTCCTTTGAAAAGTGAACGAGC-3′ designed based on L. interrogans serovar Copenhageni genome sequences (GenBank accession YP_003205). PCR was performed with KOD plus ver. 2 PCR kit (Toyobo, Osaka, Japan) from strain K64. Cycling conditions were: 95°C, 5 min, followed by 40 cycles at 95°C, 1 min, 50°C, 1 min, 68°C, 2 min, and a final extension cycle of 5 min, 68°C. PCR product was digested with NdeI and XhoI (Roche, Basel, Schweiz), ligated to NdeI- and XhoI- digested expression vector, pET-28a (+) (Novagen, San Diego, CA). The ligated plasmid was amplified in E. coli DH5α and purified using Midi PlusTM Ultrapure Plasmid Extraction System (Viogene, Taipei, Taiwan). After confirming the presence of correct inserts by sequence analysis, the plasmid was transformed in E. coli (DE3). Cultures were grown to OD₆₀₀ = 0.5 and protein expression was induced with 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG), and incubated at 25°C overnight. His-tagged LIC13300 recombinant protein (rHADH) was purified under native conditions with TALON® Metal Affinity Resin (Clontech) as previously described [59 (link)].