Composite
Part:BBa_K3105680
Designed by: Jonas Gockel Group: iGEM19_Uppsala_Universitet (2019-10-12)
Expression construct AAO-2A-eGFP
Expression circuit for aryl-alcohol oxidase (AAO) and eGFP in Pichia pastoris. AAO will be secreted due to the addition of the α-factor secretion signal and eGFP will remain in the cell. The 2A-selfcleaving peptide will lead to cleavage of the polypeptide, achieving separation of AAO from eGFP.
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1356
Illegal BamHI site found at 1543
Illegal XhoI site found at 1183 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 2503
Illegal AgeI site found at 3896 - 1000COMPATIBLE WITH RFC[1000]
Characterization
The constructs was transformed in Pichia pastoris and colonies were screened on BMMY-agar plates (contain methanol for induction of expression) (Figure 1). Visualisation of eGFP by excitation with blue light shows eGFP positive clones. Those clones are logically also expressing aryl-alcohol oxidase, as described in the part description.
Figure 1: eGFP coexpression in 2A-constructs
Transformed P. pastoris cells coexpressing eGFP and AAO were screened on a BMMY agar plate and eGFP was visualised on a blue light box under an orange filter. eGFP activity is visible for two of the restreaked colonies, which are transformed with pPICZαB_AAO-2A-eGFP. Picture was taken after 4 days of incubation at 28 °C after daily addition of 50 µL methanol on the plate lid.
Transformed P. pastoris cells coexpressing eGFP and AAO were screened on a BMMY agar plate and eGFP was visualised on a blue light box under an orange filter. eGFP activity is visible for two of the restreaked colonies, which are transformed with pPICZαB_AAO-2A-eGFP. Picture was taken after 4 days of incubation at 28 °C after daily addition of 50 µL methanol on the plate lid.
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