Part:BBa_K5443030:Experience

Our LC-MS analysis of E.coli DH10B(pMQ1C-11) cultures growing in tyrosine-enriched (0.5 g/L) LB medium showed the presence of vanillin (0.2 ppm) in reactions, and thus we can conclude that the enzyme Parts in the plasmid pMQ3C-11 were all at least partially functional. See LC-MS data below (Fig. 1).

Some preliminary evidence for successful expression of at least the C3H-HpaBC enzyme in pMQ3C comes from the brown diffusible pigment produced by E.coli DH10B(pMQ3C) cultures expressing these genes (Fig.2 - see plates in middle three columns); we believe this is a melanin-like pigment made from 'accidental' hydroxylation of tyrosine, which yields L-DOPA, which polymerises. There is precedent for this brown pigment in studies of C3H-HpaBC also elsewhere (BBa_K1124011); the E.coli C3H-HpaBC enzyme seems to be more active in making the pigment than the Saccharothrix C3H-Sam5 enzyme (in pMQ3A clones, first column in Fig.1).

Figure 1. LCMS analysis of compounds produced using pMQ3C-11.
The graph shows the detected presence of all 6 tested compounds, validating successful expression of all parts within pMQ3C-11.

Figure 2. Transformation plates of plasmids into E.coli DH10B and RARE
Some plates show diffusion of a brown pigment from colonies into the surrounding agar, suggesting a side-reaction of the enzyme C3H that produces the brown pigment L-DOPA.

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