Part:BBa_K3196033
FLO10-αpro-Geneart Laccase
This part is an improvement of part Baa_K500002, which codes the Geneart Laccase. Geneart Laccase is a polyphenol oxidase, which belongs to the family of blue multicopper oxidases. Laccases oxidize a broad range of substrates, preferably phenolic compounds.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 349
Illegal BglII site found at 886
Illegal BglII site found at 1582 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Characterization
This part is an improvement of part Baa_K500002, which codes the Geneart Laccase. Geneart Laccase is a polyphenol oxidase, which belongs to the family of blue multicopper oxidases. Laccases oxidize a broad range of substrates, preferably phenolic compounds. Compering with part BBa_K500002, We add a productive signal peptide called FLO10-αpro before the gene.
Experiments
We insert the segment into pPIC9K plasmid.
By using BamHⅠ and SnaBⅠ,we make a kick on the FLO10-pPIC9k and replace the FLO10 signal peptide with BBa_K500002 fragment(the fragment is amplified by PCR). By using SnaBⅠand EcoRⅠ, we add BBa_K500002 fragment after FLO10 signal peptide.
Following is the DNA gel electrophoresis result of construction.
Result
We transferred the constructed plasmids to Pichia Pastoris and cultured them under the same conditions for 5 days (which was the best time for expression in our main program). We measured the activity of the enzyme in a standard system. But we don’t obtain the enzyme activity data of this two part. Partly because the Geneart Laccase is wrongly folded in the Pichia pastoris GS115. Instead we break the cell by repeated freezing and thawing of liquid nitrogen and run the SDS-PAGE. Both of the constructed yeasts are cultured to the OD2.0, and treated under the same condition. Here we can find the difference in protein content:
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