Difference between revisions of "Part:BBa K3105680"
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<partinfo>BBa_K3105680 SequenceAndFeatures</partinfo> | <partinfo>BBa_K3105680 SequenceAndFeatures</partinfo> | ||
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The constructs was transformed in <I>Pichia pastoris</I> 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. | The constructs was transformed in <I>Pichia pastoris</I> 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. | ||
| − | [[File:T--Uppsala_Universitet--eGFP-plate.png|700px|thumb|left|<b>Figure 1: eGFP coexpression | + | [[File:T--Uppsala_Universitet--eGFP-plate.png|700px|thumb|left|<b>Figure 1: eGFP coexpression from 2A-constructs</b> <br> |
| − | Transformed P. pastoris cells coexpressing eGFP and AAO were screened on a BMMY agar plate | + | Transformed <I>P. pastoris</I> 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.]] |
Revision as of 14:05, 20 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.
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. Those clones are logically also expressing aryl-alcohol oxidase, as described in the part description.
Figure 1: eGFP coexpression from 2A-constructs
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.