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Pipeline publication

[…] imeSTAR DNA polymerase (Takara Bio). The PCR products were purified and cloned into the pEASY-T3 cloning vector (TransGen Biotech, Beijing, China), transformed into Escherichia coli Trans5α cells (TransGen Biotech), and then cultured in Luria-Bertani (LB) medium at 37 °C in the dark. Positive clones were sequenced. The full-length cDNA of SmKO was cloned previously., The SmCPSent, SmKS and SmKO sequences were confirmed at NCBI (http://www.ncbi.nlm.nih.gov/). The open reading frames (ORFs) and deduced amino acid sequences were analyzed using the online tool ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) and the ExPASy online tool (http://web.expasy.org/translate/), respectively. The ChloroP 1.1 Server (http://www.cbs.dtu.dk/services/ChloroP/) was used to predict chloroplast transit peptides. The sequences of the SmCPSent, SmKS and SmKO as well as other corresponding sequences downloaded from GenBank were aligned using the DNAMAN program, and the phylogenetic trees for SmCPSent, SmKS and SmKO were constructed using sequences from other plants () using the neighbor-joining method in MEGA5.1 ., The SmCPSent and SmKS ORFs (alone or in combination) were subcloned into the yeast epitope-tagging vector pESC-Trp under the control of the GAL1 or GAL10 inducible promoter (Agilent Technologies, USA) via digestion by the corresponding restriction endonucleases. The resulting constructs were verified by complete gene sequencing and then transformed into the yeast strain BY-T20 (BY4742, ΔTrp1, Trp1::HIS3-PPGK1-BTS1/ERG20-TADH1-PTDH3-SaGGPS-TTPI1-PTEF1-tHMG1-TCYC1, provided by Prof. Xueli Zhang’s lab, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, China). Then, the recombinant strains SGH1 (containing the plasmid pESC-Trp::SmCPSent), SGH2 (containing the […]

Pipeline specifications

Software tools ChloroP, DNAMAN, MEGA