Part:BBa_K5443030:Experience

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. 1 - 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. Patched clones of plasmid variants.
Each plate shows patched colonies transformed with variants of our created plasmids pMQ3B and pMQ3C. Some patched colonies show diffusion of a brown pigment into the surrounding agar, suggesting a side-reaction of the enzyme C3H that produces the brown pigment L-DOPA.

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).

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.