Difference between revisions of "Part:BBa K3105680"
 
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Expression circuit for aryl-alcohol oxidase (AAO) and eGFP in Pichia pastoris. AAO will be secreted due to the addition of the &#945;-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.
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Expression circuit for aryl-alcohol oxidase (AAO) and eGFP in <I>Pichia pastoris</I>. AAO will be secreted due to the addition of the &#945;-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.
 
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===Characterization===
 
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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.
 
  
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[[File:T--Uppsala_Universitet--eGFP-plate.png|700px|thumb|left|<b>Figure 1: eGFP coexpression in 2A-constructs</b> <br>
 
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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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<span class='h3bb'>Sequence and Features</span>
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<span class='h3bb'><b>Sequence and Features</b></span>
 
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<partinfo>BBa_K3105680 SequenceAndFeatures</partinfo>
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===Results===
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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. Due to the nature of the circuit, those clones also express AAO. <br>
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[[File:T--Uppsala_Universitet--eGFP-plate.png|700px|thumb|left|<b>Figure 1: eGFP coexpression</b> <br>
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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.]]
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Latest revision as of 14:07, 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:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1356
    Illegal BamHI site found at 1543
    Illegal XhoI site found at 1183
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 2503
    Illegal AgeI site found at 3896
  • 1000
    COMPATIBLE 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.