Difference between revisions of "Part:BBa K500001"
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To express <partinfo>BBa_K500001</partinfo>, overhang PCR was performed and the gene was cloned into the commercial 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 <I>S. cerevisiae</I>. MnP was expressed in the X-33 wildtype strain of <I>Pichia pastoris</I>, where secretion was observed (Figure 1).
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To express <partinfo>BBa_K500001</partinfo>, overhang PCR was performed to add complementary regions towards the commercial pPICZαB vector, in which MnP was cloned 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 <I>S. cerevisiae</I>. MnP was expressed in the X-33 wildtype strain of <I>Pichia pastoris</I>, where secretion was observed (Figure 1).
  
 
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Revision as of 13:50, 18 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
Author: Jonas Gockel
Summary: We studied the expression of BBa_K500001 in Pichia pastoris under the control of the AOX1 promoter BBa_K3105675 and an N-terminal fused α-secretion factor BBa_K3105674.


To express BBa_K500001, overhang PCR was performed to add complementary regions towards the commercial pPICZαB vector, in which MnP was cloned 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]