Difference between revisions of "Part:BBa K500001"
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Yeast codon optimization ,  no terminator codon,  from Phanerochaete chrysosporium.  Synthetized by Geneart  
 
Yeast codon optimization ,  no terminator codon,  from Phanerochaete chrysosporium.  Synthetized by Geneart  
 
Mn peroxidases (MnP) are extracellular hemeproteins first discovered in the white-rot fungus Phanerochaete chrysosporium, and have been virtually detected in all lignin degrading fungi so far studied. The catalytic  cycle of MnP is similar to that of other plant and fungal peroxidases
 
Mn peroxidases (MnP) are extracellular hemeproteins first discovered in the white-rot fungus Phanerochaete chrysosporium, and have been virtually detected in all lignin degrading fungi so far studied. The catalytic  cycle of MnP is similar to that of other plant and fungal peroxidases
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===Contribution===
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Group: iGEM19_Uppsala_Universitet
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<br>
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The iGEM Team Uppsala 2019 used this part to express MnP in Pichia pastoris. For this purpose, overhang PCR was performed and the gene was cloned into the pPICZαB vector by gibson assembly. In this vector, the gene is controlled by the AOX1-promoter and is cloned in-frame to an N-terminal α-secretion factor from S. cerevisiae. MnP was expressed in the X-33 wildtype strain of Pichia pastoris, where secretion was observed (Figure 1).
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The full expression circuit can be found here:
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</html> [https://parts.igem.org/wiki/index.php?title=Part:BBa_K3105682 BBa_K3105682] <html>
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</html>
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[[File:T--Uppsala_Universitet--MnP-Gel-including-legend.png|800px|thumb|left|<b>Figure 1: Expression and secretion of Manganese Peroxidase (MnP) in <I>Pichia pastoris</I> </b> <br> X-33 <I>P. pastoris</I> cells were transformed with pPICZαB_MnP and expression cultures were induced. Different fractions (pellet and supernatant samples / uninduced and induced cultures) from X-33 MnP expression culture were analysed on a 10 % SDS-PAGE stained with Coomassie Blue. Induction bands can be seen at a size of approximately 70 kDa in the induced samples for both pellet and supernatant fraction (red arrowhead), showing a secretion of MnP.]]
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 19:13, 17 October 2019

lignin degradation 2

Yeast codon optimization , no terminator codon, from Phanerochaete chrysosporium. Synthetized by Geneart Mn peroxidases (MnP) are extracellular hemeproteins first discovered in the white-rot fungus Phanerochaete chrysosporium, and have been virtually detected in all lignin degrading fungi so far studied. The catalytic cycle of MnP is similar to that of other plant and fungal peroxidases



Contribution

Group: iGEM19_Uppsala_Universitet

The iGEM Team Uppsala 2019 used this part to express MnP in Pichia pastoris. For this purpose, overhang PCR was performed and the gene was cloned into the pPICZαB vector by gibson assembly. In this vector, the gene is controlled by the AOX1-promoter and is cloned in-frame to an N-terminal α-secretion factor from S. cerevisiae. MnP was expressed in the X-33 wildtype strain of Pichia pastoris, where secretion was observed (Figure 1).


The full expression circuit can be found here: BBa_K3105682

Figure 1: Expression and secretion of Manganese Peroxidase (MnP) in Pichia pastoris
X-33 P. pastoris cells were transformed with pPICZαB_MnP and expression cultures were induced. Different fractions (pellet and supernatant samples / uninduced and induced cultures) from X-33 MnP expression culture were analysed on a 10 % SDS-PAGE stained with Coomassie Blue. Induction bands can be seen at a size of approximately 70 kDa in the induced samples for both pellet and supernatant fraction (red arrowhead), showing a secretion of MnP.




























Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 220
    Illegal BglII site found at 512
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]