Difference between revisions of "Part:BBa K3196013"
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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.
−
[[File:T--HUST--China--2019-PHO1SLAC.jpg ‎|525px|thumb|center|Figure1. AOX1-FLO10-SLAC-His tag.]]
+
[[File:T--HUST--China--2019-PHO1SLAC.jpg ‎|525px|thumb|center|Figure1. AOX1-PHO1-SLAC-His tag.]]
  
 
<h1>'''DNA Gel Electrophoretic'''</h1>
 
<h1>'''DNA Gel Electrophoretic'''</h1>
−
After we link FLO10 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 1989 bp which means the PCR is successful.
+
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 1989 bp which means the PCR is successful.
 
[[File:T--HUST-China--2019-DNA Gel Electrophoretic.png|400px|thumb|center|Figure1:This is the result of the SacⅠ single endonuclease digestion plasmid of the TOP10 strain, we will extract the gel and obtain the aimed DNA. ]]
 
[[File:T--HUST-China--2019-DNA Gel Electrophoretic.png|400px|thumb|center|Figure1:This is the result of the SacⅠ single endonuclease digestion plasmid of the TOP10 strain, we will extract the gel and obtain the aimed DNA. ]]
  
 
<h1>'''SDS-PAGE'''</h1>
 
<h1>'''SDS-PAGE'''</h1>
−
We run the SDS-PAGE to check whether FLO10 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.
+
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.
 
[[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.]]
 
[[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.]]
  

Revision as of 08:11, 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.

DNA Gel Electrophoretic

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 1989 bp which means the PCR is successful.

Figure1:This is the result of the SacⅠ single endonuclease digestion plasmid of the TOP10 strain, we will extract the gel and obtain the aimed DNA.

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