Difference between revisions of "Part:BBa K5443003:Experience"

Latest revision as of 11:51, 2 October 2024

We succeeded in producing a relatively large amount of caffeate (35 ppm) relative to the other metabolites, and we also made a small amount of vanillin (0.2 ppm), and thus we can conclude that the enzyme Parts in the plasmid pMQ3C-11 were all at least partially functional, including the 4-Coumarate 3-Hydroxylase C3H-HpaBC. See LC-MS data below (Fig. 1).

Further preliminary evidence for successful expression of the C3H-HpaBC enzyme comes from the brown diffusible pigment produced by E.coli cultures expressing these genes (Fig. 2); we believe this is a melanin-like pigment made from tyrosine via L-DOPA as an intermediate. There is precedent for this with previous work with HpaBC (BBa_K1124011).

**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. 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 HpaBC that produces the brown pigment L-DOPA.

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-===Applications of BBa_K5443003===
+We succeeded in producing a relatively large amount of caffeate (35 ppm) relative to the other metabolites, and we also made a small amount of vanillin (0.2 ppm), and thus we can conclude that the enzyme Parts in the plasmid pMQ3C-11 were all at least partially functional, including the 4-Coumarate 3-Hydroxylase C3H-HpaBC. See LC-MS data below (Fig. 1).
-We showed that the HpaBC enzyme was functional in <i>E. coli</i> as part of a vanillin biosynthetic pathway, as evidenced by production of caffeate (see LC-MS data from one clone pMQ3C-11, Fig. 1). Further evidence for functional expression of the HpaBC monooxygenase was seen from production of a brown melanin-type dye as a side-reaction (see Fig. 2); we believe this indicates that HpaBC is acting on tyrosine (to make L-DOPA, which polymerises) in addition to our intended reaction on 4-coumarate to make caffeate.
+Further preliminary evidence for successful expression of the C3H-HpaBC enzyme comes from the brown diffusible pigment produced by E.coli cultures expressing these genes (Fig. 2); we believe this is a melanin-like pigment made from tyrosine via L-DOPA as an intermediate. There is precedent for this with previous work with HpaBC (BBa_K1124011).