Difference between revisions of "Part:BBa K5443003:Experience"
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===Applications of BBa_K5443003===
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We succeeded in producing a relatively large amount of caffeate (35 ppm) relative to the other metabolites, 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).  
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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.  
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Revision as of 11:26, 2 October 2024

We succeeded in producing a relatively large amount of caffeate (35 ppm) relative to the other metabolites, 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).


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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