Cells containing plasmids with Part:BBa_K323135 and Part:BBa_K323132 were transformed with Part:BBa_K323066, Part:BBa_K323153 and a commercial pBluescript plasmid without a DNA program. Cultures were incubated at 30°C for several days and samples were taken at various times. Violacein was extraced from individual samples to analyze the products and evaluate the yield of violacein production.
Figure: Bacterial cultures carrying plasmids containing a DNA program, a scrambled DNA program and no DNA program. Cultures of bacteria carrying plasmids with
Part:BBa_K323132,
Part:BBa_K323135 and i) plasmid containing a DNA program
Part:BBa_K323066, ii) plasmid containing a scrambled DNA program
Part:BBa_K323153 and iii) pBluescript plasmid without a DNA program were incubated at 30°C in a Luria Bertani media. The color of the culture containing the plasmid with the DNA program is notably more purple than the color of other two cultures.
Figure: Violacein extracts of bacterial cultures carrying plasmids containing a DNA program, a scrambled DNA program and no DNA program. Violacein was extracted from the incubated cultures. An extensive difference in color intensity is visible between the extracts of all three cultures, among which the extract of the culture containing a plasmid with the DNA program exhibits the most intensive purple color.
By measuring the concentrations of violacein in the extracts of taken samples, we determined that the speed and yield of violacein production in our system increased 6-fold in comparison to an E. coli strain containing plasmids with a scrambled DNA program or without a DNA program.
Figure: Violacein concentrations measured at various times through the experiment. The results above show that cultures containing the DNA program produce violacein more rapidly and efficiently.
Figure: Violacein concentrations at the end of the experiment. Measuring the concentration of violacein in extracts of the incubated cultures determined the highest yield of violacein in the culture with the DNA program. The production was increased 6-fold in comparison with the culture without a DNA program. A significant disctintion from the culture with the scrambled program is also notable, which implies that a correct arrangement of enzymes on the DNA program is important for the speed of the reaction.
In addition, the 2010 iGEM team Slovenia have shown that a DNA program-mediated biosynthesis decreases formation of an unwanted side product deoxychromoviridans. We have analysed the products of the vio operon (Part:Bba_K274002) with HPLC, TLC and mass spectrometry (for detailed description see the 2010 iGEM team Slovenia [http://2010.igem.org/Team:Slovenia/METHODS_and_PARTS wiki]).
Figure: Silica gel plate of culture extracts. The extract of the culture without a DNA program displayed three bands: a violacein, a deoxyviolacein and a deoxychromoviridans band. The extract of the culture containing the DNA program contained only violacein and small amounts of deoxyviolacein, while no deoxychromoviridans could be detected in the sample.
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