Difference between revisions of "Part:BBa K3105679"
Line 3: Line 3:
 
<partinfo>BBa_K3105679 short</partinfo>
 
<partinfo>BBa_K3105679 short</partinfo>
  
−
Expression circuit for HRP and AAO in Pichia pastoris. HRP will be secreted due to the addition of the &#945;-factor secretion signal and AAO will remain in the cell. The 2A-selfcleaving peptide will lead to cleavage of the polypeptide, achieving separation of HRP from AAO.
+
Expression circuit for HRP and AAO in Pichia pastoris. HRP will be secreted due to the addition of the &#945;-factor secretion signal and AAO will remain in the cell. The 2A-selfcleaving peptide will lead to cleavage of the polypeptide, achieving separation of HRP from AAO. <br><br>
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
Line 9: Line 9:
  
 
<!-- -->
 
<!-- -->
−
<span class='h3bb'>Sequence and Features</span>
+
<span class='h3bb'><b>Sequence and Features</b></span>
 
<partinfo>BBa_K3105679 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3105679 SequenceAndFeatures</partinfo>
 +
 +
 +
===Results===
 +
<div>
 +
The part was expressed in X-33 <I>P. pastoris</I> cells using the pPICZαB shuttle vector. Cultures were induced with methanol and analysed on SDS-PAGE. HRP and AAO was successfully expressed (Figure 1). Furthermore, also the functionality of the 2A-peptide in <I>P. patoris</I> was shown, because failure in cleavage would lead to a induction band bigger than 100 kDa.
 +
<br><br>
 +
[[File:T--Uppsala Universitet--HRP-2A-AAO Gel.png|800px|thumb|left|<b>Figure 1: Expression of HRP-2A-AAO</b><br>
 +
X-33 <I>P. pastoris</I> cells were transformed with pPICZαB_HRP-2A-AAO and expression cultures were induced. Different fractions (pellet (P) and supernatant (S) samples / uninduced (u) and induced (i) cultures) from X-33 HRP-2A-AAO expression culture were analysed on a 10 % SDS-PAGE stained with Coomassie Blue. After 24 h an induction band can be seen at around 55 kDa, which is approximately the calculated molecular weight for both HRP and AAO. This shows that the enzymes are expressed as well as that the cleavage initiated by the 2A-peptide is functional.]]
 +
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
 +
Enzyme activity was assessed by conducting different assays. For HRP activity, an ABTS-assay was used and the activity between uninduced and induced samples was compared (Figure 2A). Clear increase
  
  

Revision as of 13:11, 20 October 2019


Expression construct HRP-2A-AAO

Expression circuit for HRP and AAO in Pichia pastoris. HRP will be secreted due to the addition of the α-factor secretion signal and AAO will remain in the cell. The 2A-selfcleaving peptide will lead to cleavage of the polypeptide, achieving separation of HRP from AAO.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2086
    Illegal BamHI site found at 2481
    Illegal BamHI site found at 2668
    Illegal XhoI site found at 1183
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 2167
    Illegal AgeI site found at 3628
    Illegal AgeI site found at 4241
  • 1000
    COMPATIBLE WITH RFC[1000]


Results

The part was expressed in X-33 P. pastoris cells using the pPICZαB shuttle vector. Cultures were induced with methanol and analysed on SDS-PAGE. HRP and AAO was successfully expressed (Figure 1). Furthermore, also the functionality of the 2A-peptide in P. patoris was shown, because failure in cleavage would lead to a induction band bigger than 100 kDa.

Figure 1: Expression of HRP-2A-AAO
X-33 P. pastoris cells were transformed with pPICZαB_HRP-2A-AAO and expression cultures were induced. Different fractions (pellet (P) and supernatant (S) samples / uninduced (u) and induced (i) cultures) from X-33 HRP-2A-AAO expression culture were analysed on a 10 % SDS-PAGE stained with Coomassie Blue. After 24 h an induction band can be seen at around 55 kDa, which is approximately the calculated molecular weight for both HRP and AAO. This shows that the enzymes are expressed as well as that the cleavage initiated by the 2A-peptide is functional.


































Enzyme activity was assessed by conducting different assays. For HRP activity, an ABTS-assay was used and the activity between uninduced and induced samples was compared (Figure 2A). Clear increase