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Team:TU Darmstadt/Notebook/Labjournal/K1497010
From 2014.igem.org
K1497010 - Dihydroflavonol 4-reductase (DFR)
The dihydroflavonol 4-reductase from Dianthus caryophyllus (dcDFR) is the first enzyme in the anthocyanidine biosynthesis. It catalyses the reduction of dihydroflavonols to their corresponding leucoanthocyanidines.
dcDFR was kindly received from Dr. Stefan Martens (Research and Innovation Centre, Fondazione Edmund Mach, Italy) in the yeast shuttle vector pYES2. The gene of dcDFR is composed of 1062 base pairs and has two EcoRI restriction sites at position 577 and 622. To use the standard iGEM biobrick prefix and suffix for molecular cloning, the restriction sites had to be removed. This was achieved by site-directed mutagenesis with the primers DFR_mut_EcoRI_577_F, DFR_mut_EcoRI_577_R, DFR_mut2_EcoRI_622_F and DFR_mut2_EcoRI_622_R.
Figure 1: Restriction test of mutated dcDFR, 2nd lane contains dcDFR not digested (green box), 3rd lane contains dcDFR digested with EcoRI (red box)
After site-directed mutagenesis DFR was still in the yeast shuttle vector pYES2. To transform DFR into pSB1C3, we first added Prefix and Suffix via PCR with the Prefix _DFR -primer and the DFR_Suffix-primer.
Figure 2: Agarose gel electrophoresis after PCR: 3rd and 4th lane (both red box) contains dcDFR with prefix and suffix
After addition of prefix and suffix DFR was digested with the restriction enzymes EcoRI and PstI and ligated into an equally digested pSB1C3 vector to create the biobrick BBa_K1497010.
