Composite
Part:BBa_K3105682
Designed by: Jonas Gockel Group: iGEM19_Uppsala_Universitet (2019-10-12)
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:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1438
Illegal BglII site found at 1730
Illegal XhoI site found at 1189 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 2491
- 1000COMPATIBLE WITH RFC[1000]
Characterization
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.
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.
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