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]
Results
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. Due to the nature of the circuit, those clones also express AAO.
Figure 1: eGFP coexpression
Transformed P. pastoris cells coexpressing eGFP and AAO were screened on a BMMY agar plate. eGFP is visible for two of the restreaked colonies, in the middle. Picture was taken on a blue light box under an orange filter after 4 days of incubation at 28 °C with 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. eGFP is visible for two of the restreaked colonies, in the middle. Picture was taken on a blue light box under an orange filter after 4 days of incubation at 28 °C with daily addition of 50 µL methanol on the plate lid.
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