Chemistry and Microbiology
The goal of this thesis is to determine the conditions that give the highest yield of indigo and indirubin in recombinant E.coli. This E.coli has been transformed with a P450 gene from woad, an indigo producing plant. The conditions investigated were the type of media, the amount of inducer, the presences of supplements, and the induction time. The techniques used during the experiments were liquid-liquid extraction using separatory funnels and centrifugation, rotary evaporation, UV-vis spectrophotometry, and thin layer chromatography. Thin layer chromatography showed that the products of the reaction are indigo and indirubin; UV-vis spectrophotometry was used to find the absorbance at different wavelengths, which were used to calculate the concentration of indigo and indirubin in each sample, and determine the conditions that gave the highest yield. These conditions were found to be TB for the medium, 40 µL of IPTG, the presence of supplements, and 96 hours for the induction time.