Difference between revisions of "Part:BBa K3105682"
 
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===Characterization===  
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===Results===  
 
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The part was expressed in X-33 <I>P. pastoris</I> cells using the pPICZ&#945;B shuttle vector. Cultures were induced with methanol and analysed on SDS-PAGE. MnP was successfully expressed and secreted (Figure 1).
 
The part was expressed in X-33 <I>P. pastoris</I> cells using the pPICZ&#945;B shuttle vector. Cultures were induced with methanol and analysed on SDS-PAGE. MnP was successfully expressed and secreted (Figure 1).

Latest revision as of 00:20, 20 October 2019


Expression construct MnP

Manganese Peroxidase (MnP) cloned in-frame with α-factor from Saccharomyces cerevisiae. This construct is controlled by the AOX1 promotor, which is inducible by methanol.


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 1438
    Illegal BglII site found at 1730
    Illegal XhoI site found at 1189
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 2491
  • 1000
    COMPATIBLE WITH RFC[1000]



Results

The part was expressed in X-33 P. pastoris cells using the pPICZαB shuttle vector. Cultures were induced with methanol and analysed on SDS-PAGE. MnP was successfully expressed and secreted (Figure 1).


Figure 1: Expression and secretion of Manganese Peroxidase (MnP)
X-33 P. pastoris cells were transformed with pPICZαB_MnP, induced (i), fractionated into pellets (P) and supernatants (S), and analysed by 10 % SDS-PAGE stained with Coomassie Blue. u, uninduced
Induction bands are visible at approximately 70 kDa for both P and S (red arrows), showing secretion of MnP.