Ligation solution 1

The strain L. brevis ATCC 367 was obtained from the Institute of Microbiology of the Chinese Academy of Sciences. The strain K. pneumoniae DSM2026 was obtained from Doctor An-Ping Zeng (Hamburg University of Technology). E. coli DH5α was used as host strains for cloning. BL21 (DE3) was used as host strains for expression. Plasmid pET-32a(+) was employed as an expression vector.
All enzymes, such as restriction endonucleases, T4 DNA ligase and Ex Taq DNA, were recruited from TaKaRa Co., Ltd. (Dalian, China). PrimeSTAR HS DNA Polymerase, Ligation solution I, Agarose Gel DNA Purification Kit Ver 2.0, Mutan BEST Kit, Agarose Gel DNA Fragment Recovery Kit Ver.2.0 and pMD18-T were obtained from TaKaRa Co., Ltd. (Dalian, China). GeneRuler Ladder Mix was purchased from MBI Co. All other chemicals used were analytically graded and were purchased from either Sigma China or Omiga China.

A double mutant was constructed through site-directed mutagenesis. First, the recombinant plasmid was amplified with PCR using mutagenic oligonucleotides (see Table 1) and the MutanBEST Kit (TaKaRa). Then, the amplified fragments were purified and isolated from 0.8% (w/v) agarose gels after electrophoresis. The fragment was blunted with Blunting Kination Enzyme Mix (TaKaRa). The blunt-end fragment was ligated with Ligation Solution I (TaKaRa). The competent cells of E. coli JM109 were then transformed by the reaction mixture. Selections of the transformants were performed on LB agar plates containing 50 µg/mL ampicillin. l-AAD in the transformant was confirmed with PCR and checked through sequencing, and the recombinant plasmids were finally transformed into competent cells of E. coli BL21 (DE3) for expression.