Difference between revisions of "Team:Uppsala/Zea-Strain"

After creating the zeaxanthin producing strain, we combined it with the plasmid containing the extended crocin pathway which gave us an E. coli strain including the entire production pathway from FPP to crocin. The three enzyme BioBricks BBa_K2423005, BBa_K2423007 and BBa_K2423008 in the zeaxanthin-crocin pathway were assembled to one plasmid (pSB1A3) using 3A assembly and was inserted into the zeaxanthin producing E.coli strain using electroporation. The resulting plate can be seen in figure 5.

The color of the colonies changes slightly at each addition of another enzyme construct (another step in the crocin pathway). This is an indication that something is indeed happening with the bacterial pigment production when we introduce our pathway steps (figure 6) into the zeaxanthin strain. In the future it would be good to integrate the whole pathway from farnesyl pyrophosphate (FPP) to crocin into the chromosome for a stable crocin producing strain that does not require antibiotic selection which would make it easier to use as for example food coloring.

We wanted to analyze the compounds found after extraction of the zeaxanthin strains with and without plasmids from the crocin pathway. Thin layer chromatography (TLC) was used to do this on a zeaxanthin strain, a zeaxanthin strain containing CaCCD2 (BBa_K2423005), a zeaxanthin strain containing a plasmid with both CaCCD2 and CsADH2946, standards for crocetin and crocetin dialdehyde and a wild-type E. coli strain. As you can see from the resulting TLC plate in figure 7, many different pigments (represented by the bands) are present in the extract from the zeaxanthin strain. No pigment bands are present in the extract from the negative control strain (wild type E. coli), showing that the pigments in the zeaxanthin strain are precursors to zeaxanthin and zeaxanthin itself. This is consistent with the absorbance measurement where zeaxanthin was seen to be present in the extract (Figure 4.). Multiple pigments are also formed by the zeaxanthin + CaCCD2 and zeaxanthin + CaCCD2 + CsADH2946 strains (Figure 7.). Due to the mix of pathway pigments found in the samples, and limitations in resolution in the TLC, identification of specific pigment cannot be made from this data. In the future it would be very interesting to uniquelly identify the identity of the different pigments formed by our strains using a more informative method such as High-performance liquid chromatography-mass spectrometry (HPLC-MS).