T4 dna ligase and restriction endonucleases

T4 DNA ligase and restriction endonucleases were obtained from New England Biolabs. DNA Polymerase Mastermix was from Denville Scientific. Ni-NTA Superflow, QIAprep Miniprep kits, and QIAquick gel extraction kits were purchased from Qiagen. All reagents and cofactors were from Sigma-Aldrich or Thermo Fisher Scientific. Oligonucleotide primers were synthesized by Valuegene and IDT. Gene sequencing and gene synthesis were performed by Genewiz and Twist Bioscience. Assembly master mix (AMM) used for cloning was prepared as outlined in^{19 (link)}. All DNA and protein concentrations were measured with a Thermo Fisher Scientific Nanodrop 1000 Spectrophotometer. Vials for small scale reactions, for gas chromatography and 96-well plates were purchased from VWR. Viton tubing was purchased from Cole-Parmer Instrument Company. Colorimetric enzyme-coupled assays were performed in 96-well plates and measured with Molecular Devices SpectraMax M5 microplate reader. When performing large numbers of assays simultaneously, Integra Viafill and Integra Viaflo instruments were used for reagent additions. Colonies for screening of mutant variants were selected automatically using a Molecular Devices Qpix 420 colony picker.

T4 DNA ligase and restriction endonucleases were obtained from New England Biolabs. DNA Polymerase Mastermix was from Denville Scientific. Ni-NTA Superflow, QIAprep Miniprep kits and QIAquick gel extraction kits were purchased from Qiagen. The λDE3 Lysogenization Kit was from EMD Chemicals. All reagents were from Sigma Aldrich except for LB agar and Terrific Broth which were obtained from Fisher Scientific. Oligonucleotide primers were synthesized by Valuegene and IDT. Gene sequencing and gene synthesis were performed by Genewiz. Assembly master mix (AMM) used for cloning was prepared as outlined in43 (link). All DNA and protein concentrations were measured with Thermo Fisher Scientific Nanodrop 1000 Spectrophotometer. Colorimetric enzyme-coupled assays were performed in 96-well plates and measured with Molecular Devices SpectraMax M5 microplate reader.

Erythrobacter seohaensis SW-135 was kindly provided by Prof. Jung-Hoon Yoon and cultivated in marine broth 2216 (BD Difco^TM, United States) at 30°C (Yoon et al., 2005 (link)). Kits for genomic DNA isolation, DNA purification, and plasmid isolation were purchased from Omega (United States). DNA polymerase was purchased from TaKaRa (China) and T4 DNA ligase and restriction endonucleases were purchased from New England Biolabs (United States). Plasmid pSMT3 (Herrmann et al., 1996 (link)) was stored in our lab and used as the vector for gene cloning and sequencing as well as protein expression. Escherichia coli BL21 (DE3) was used for protein expression. E. coli strains were grown at 37°C in Lysogeny broth (LB) medium containing 10 g/L NaCl, 10 g/L tryptone, and 5 g/L yeast extract (BD Difco^TM, United States), pH 7.0, supplemented with kanamycin (50 μg/mL) when required. Ni Sepharose (GE Healthcare, United States) was used to purify the His₆-tagged protein. p-Nitrophenyl (NP) acetate (C2), p-NP butyrate (C4), p-NP caprylate (C8), p-NP decanoate (C10), p-NP laurate (C12), p-NP myristate (C14), and p-NP palmitate (C16) were purchased from Sigma–Aldrich (United States) and p-NP hexanoate (C6) was purchased from TCI (Japan).