Puc118

Total RNA was extracted from each sample using a Qiagen RNeasy Plant Mini Kit using the suppliers’ protocols then converted to cDNA using SuperScript III reverse transcriptase (Invitrogen, Tokyo, Japan) and SBWMV-UNIR (Clover et al., 2001 (link)). TaqMan primers and probe were targeted to the JSBWMV coat protein sequence. PCRs were formulated with PrimeSTAR HS DNA Polymerase (Takara Bio inc., Kusatsu, Shiga, Japan) and the primer pair SBWMV-UNIF/SBWMV-UNIR (Clover et al., 2001 (link)). The resulting amplicon (expected size: 338 bp) was verified by separation through a 1.5% agarose gel and purified using a QIA quick PCR Purification Kit (Qiagen) following the manufacturer’s protocol. The purified amplicons were ligated into pUC118 (Takara Bio inc.) and transformed into E. coli DH5α competent cells (Invitrogen) for sequencing using a Hitachi 3130X Genetic Analyzer.

The vector pUC118, restriction endonuclease and Eschericia coli DH5α cells were purchased from TaKaRa (Dalian, China), the expression vector pET-30a (+) and E. coli BL21 (DE3) cells were purchased from Novagen (Merck, United States). His-tag-specific purification kit was purchased from GE Healthcare (Pittsburgh, PA, United States). All the chemical reagents are chemically pure.

Escherichia coli DH5α and E. coli BL21 (DE3) (Novagen, Madison, WI, USA) was used as the host for gene cloning and protein expression. pUC118 (TaKaRa) and pET28a (+) (Novagen) was used to construct metagenomic library and express the target gene.

Escherichia coli DH5α and E. coli BL21 (DE3) (Novagen, Madison, WI, United States) were used as the host for molecular cloning. The pUC118 (TaKaRa) and pET-28a (+) (Novagen) were used to construct metagenomic libraries and express the target protein, respectively.

To clone the FE-hydrolyzing carboxylesterase gene, the shotgun method was used to
construct a gene library of strain JPL-2 in E. coli DH5á.
Genomic DNA of strain JPL-2 was extracted by the method described above and
digested partially with Sau3AI. Fractions containing
approximately 4 to 6 kb DNA fragments were pooled, ligated into the
BamHI site of the plasmid pUC118 (TaKaRa Biotechnology,
Dalian, China), and transformed into competent cells of E.coliDH5|Á. The transformants were plated onto LB agar plates containing 100 mg
L⁻¹ ampicillin and 100 mg L⁻¹ FE and incubated at 37
°C for 24 h. Colonies that degraded FE produced clear transparent halos and were
screened and further tested for their degrading capabilities. Analysis of the
nucleotide and deduced amino acid sequence was performed using Omiga 2.0. BlastN
and BlastP were used for the nucleotide sequence and amino acid identity
comparison, respectively. (www.ncbi.nlm.nih.gov/Blast).

E. coli DH5α and pUC118 (TaKaRa, Dalian, China) were used for construction of metagenomic libraries, random mutagenesis libraries, and gene cloning. E. coli BL21 (DE3) and pET-32a (+) (Novagen, Madison, WI, USA) were used for protein expression. Restriction endonucleases, DNA polymerase, and T4 DNA ligase were purchased from Thermo Fisher Scientific (Hudson, NH, USA). Cellobiose, glucose, and p-nitrophenyl-β-d-glucopyranoside (pNPG) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals and reagents were of analytical grade and purchased from commercial sources, unless indicated otherwise.

To construct the PGN_1134 or PGN_1135 mutants, the downstream region of the PGN_1134 gene or upstream region of the PGN_1135 gene was amplified with PGN1134dwFw/PGN1134dwRv or PGN1135upFw/PGN1135upRv primer pairs, respectively, using P. gingivalis ATCC 33277 genome as the DNA template, and was cloned into pUC118 (TaKaRa, Kusatsu, Japan). The downstream region and upstream region were then purified using lower PstI-SacI sites or lower SphI-BamHI sites from each cloned recombinant plasmid and then exchanged with the corresponding sites of pKD990, which is PGN_1134-1135::ermF in pGEM T-easy vector (16 (link)), yielding PGN_1134::ermF or PGN_1135::ermF in pGEM T-easy vector. Finally, each desired recombinant plasmid was digested with SacI and introduced into P. gingivalis ATCC 33277 by electroporation to generate Em^r transformant, yielding PGN_1134 (KDP1107) or PGN_1135 (KDP1108) mutants. Transformants were selected on blood agar plates containing 10 μg/mL erythromycin.