Difference between revisions of "Part:BBa K2779900"

Revision as of 00:14, 18 October 2018

HemA from R. capsulatus

This gene, which was codon-optimized for E. coli, encodes for 5-aminolevulinic acid synthase from Rhodobacter capsulitis. It catalyzes the formation of 5-aminolevulinate from succinyl CoA and glycine, according to the following scheme.

Characterization=

Previous work has shown that overexpressing 5-aminolevulinic acid synthase can drive the expression of PPIX and that excess PPIX can be secreted in the media [1]. Thus, we transformed a plasmid carrying HemA under the pBAD promoter and cultured it in 250 mL of modified TB media supplemented with antibiotics and L-arabinose according to standard protocols. Using two slightly different protocols and solvent systems from previous work, we then collected the secreted porphyrins using DEAE-Sephadex A-25, an anion exchange resin. Elution of the adsorbed porphyrins from the resin allowed us to characterize the porphyrins via fluorescence and thin layer chromatography (TLC) and compare the results against that of a commercially available standard (referred to as “stock”).

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1219
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 [[File:T--UAlberta--HemARxn.png]]
+===Characterization====
+Previous work has shown that overexpressing 5-aminolevulinic acid synthase can drive the expression of PPIX and that excess PPIX can be secreted in the media [1]. Thus, we transformed a plasmid carrying HemA under the pBAD promoter and  cultured it in 250 mL of modified TB media supplemented with antibiotics and L-arabinose according to standard protocols. Using two slightly different protocols and solvent systems from previous work, we then collected the secreted porphyrins using DEAE-Sephadex A-25, an anion exchange resin. Elution of the adsorbed porphyrins from the resin allowed us to characterize the porphyrins via fluorescence and thin layer chromatography (TLC) and compare the results against that of a commercially available standard (referred to as “stock”).
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