Difference between revisions of "Part:BBa K3196013"
 
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PHO1 is one of three repressible acid phosphatases, a glycoprotein that is transported to the cell surface. SLAC can catalyze lignin.
 
PHO1 is one of three repressible acid phosphatases, a glycoprotein that is transported to the cell surface. SLAC can catalyze lignin.
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<h1>'''Characterization'''</h1>
 
<h1>'''Characterization'''</h1>
 
This is a four section for degrade and transfer lignin part.
 
This is a four section for degrade and transfer lignin part.
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[[File:T--HUST--China--2019-PHO1-αproSLAC.jpg ‎|400px|thumb|center|Figure1.T--HUST--China--2019-PHO1-αproSLAC]]
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[[File:T--HUST--China--2019-PHO1SLAC.jpg ‎|525px|thumb|center|Figure1. AOX1-PHO1-SLAC-His tag.]]
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<h1>'''DNA Gel Electrophoretic'''</h1>
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After we link PHO1 and SLAC successfully, we run the PCR with an intention to confirm the expression of SLAC. As the figure shows, we get the genetic stripe about xxx bp which means the PCR is successful.
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[[File:Kozak_sequence.png ‎|400px|thumb|center|Figure1. This figure shows the most possible kozak consensus sequence.]]
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<h1>'''SDS-PAGE'''</h1>
 
<h1>'''SDS-PAGE'''</h1>
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We run the SDS-PAGE to check whether PHO1 help the enzyme transfer to the extracellular. As the figure shows, we get the protein type about xxx bp which means the SDS-PAGE is successful.
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We run the SDS-PAGE to check whether PHO1 help the enzyme transfer to the extracellular. As the figure shows, we get the protein type about 35 KDa which means the SDS-PAGE is successful.
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[[File:Kozak_sequence.png ‎|400px|thumb|center|Figure1. This figure shows the most possible kozak consensus sequence.]]
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[[File:T--HUST-China--2019-SLAC-SDS-PAGE.jpg |400px|thumb|center|Figure3:the SDS-page result shows that Pichia pastoris have successfully secrete SLAC protein into superfluous liquid.]]
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<h1>'''Enzyme Activity'''</h1>
 
<h1>'''Enzyme Activity'''</h1>
 
We use ABTS to detect the enzyme activity. As the figure shows, the solution turns green, which confirm the enzyme activity.
 
We use ABTS to detect the enzyme activity. As the figure shows, the solution turns green, which confirm the enzyme activity.
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[[File:Kozak_sequence.png ‎|400px|thumb|center|Figure1. This figure shows the most possible kozak consensus sequence.]]
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[[File:T--HUST-China--2019-SLAC enzyme activity.png|400px|thumb|center|Figure4:these four pictures shows the enzyme activity changes with the time. The four kinds of Pichia pastoris have the different curve, and the most activity strain is FLO10-apro, PHO5apro shows a more delayed increase on enzyme activity.]]
  
 
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Latest revision as of 11:21, 20 October 2019


AOX1-Kozak-PHO1-SLAC-His tag-AOX1 Terminator

PHO1 is one of three repressible acid phosphatases, a glycoprotein that is transported to the cell surface. SLAC can catalyze lignin.

Characterization

This is a four section for degrade and transfer lignin part.

Figure1. AOX1-PHO1-SLAC-His tag.

SDS-PAGE

We run the SDS-PAGE to check whether PHO1 help the enzyme transfer to the extracellular. As the figure shows, we get the protein type about 35 KDa which means the SDS-PAGE is successful.

Figure3:the SDS-page result shows that Pichia pastoris have successfully secrete SLAC protein into superfluous liquid.


Enzyme Activity

We use ABTS to detect the enzyme activity. As the figure shows, the solution turns green, which confirm the enzyme activity.

Figure4:these four pictures shows the enzyme activity changes with the time. The four kinds of Pichia pastoris have the different curve, and the most activity strain is FLO10-apro, PHO5apro shows a more delayed increase on enzyme activity.

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 937
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1367