Before we started our experiments, we obtained a violacein standard from Sigma-Aldrich,Item:V9389-1MG . We used this standard to measure the absorbance of violacein at varying concentrations, obtaining the standard curve shown below. Full wavelength scans were obtained, but as shown by previous iGEM teams (Cambridge 2009 ) the maximum absorbance of violacein is seen at 575nm and is reported here. During our experiments we worked with several different strains of E. coli, each containing plasmids with different violacein constructs:
Strain 1: pcdfDuet+VioABE
Strain 2:pcdfDuet+VioDC
Strain 3: pcdfDuet:VioABE+VioDC (full operon)
While other teams and papers have reported that extracting pigment from cultures is the best way to perform analysis and quantification, several different solvents have been reported. The 2009 Cambridge iGEM team used acetone to extract, so we tested extraction of violacen from Strain 3 (full violacein operon) using acetone as well as methanol and ethanol.
Our results show that ethanol was the best solvent to use for extraction and so the rest of our extractions were performed in ethanol. The details of growth and sample analysis can be found on our protocols page.
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