Difference between revisions of "Part:BBa K3105680"

Revision as of 18:32, 18 October 2019

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.

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.

Sequence and Features