Difference between revisions of "Part:BBa K3196012"
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K3196012 short</partinfo>
 
<partinfo>BBa_K3196012 short</partinfo>
−
 
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FLO10 αpro is a combined signal peptide, which enhance the enzyme activity 3 times. SLAC can catalyze lignin.
 
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<h1>'''Characterization'''</h1>
 
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This is a four section for degrade and transfer lignin part.
 
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[[File:T--HUST--China--2019-FLO10-αproSLAC.jpg ‎|400px|thumb|center|Figure1. T--HUST--China--2019-FLO10-αproSLAC.]]
 
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<h1>'''DNA Gel Electrophoretic'''</h1>
 
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After we link FLO10 αpro 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>
 
−
We run the SDS-PAGE to check whether FLO10 αpro 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.
 
−
[[File:Kozak_sequence.png ‎|400px|thumb|center|Figure1. This figure shows the most possible kozak consensus sequence.]]
 
−
 
−
<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.
 
−
[[File:Kozak_sequence.png ‎|400px|thumb|center|Figure1. This figure shows the most possible kozak consensus sequence.]]
 
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<partinfo>BBa_K3196012 parameters</partinfo>
 
<partinfo>BBa_K3196012 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
The FLO10 was combined with the guiding peptide sequence of saccharomyces cerevisiae to form the signal peptide FLO10- αpro.FLO10 αpro is a combined signal peptide, which enhance the enzyme activity 3 times.
 +
 +
 +
<h1>'''Characterization'''</h1>
 +
This is a four section for degrade and transfer lignin part.
 +
[[File:T--HUST--China--2019-FLO10SLAC.jpg ‎|525px|thumb|center|Figure1. AOX1-FLO10-SLAC-His tag.]]
 +
 +
<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 2208 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. ]]
 +
 +
<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.
 +
[[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.]]
 +
 +
 +
<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.
 +
[[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.]]

Revision as of 07:14, 20 October 2019


AOX1-Kozak-FLO10 pro-SLAC-His tag-AOX1 Terminator

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 1586


The FLO10 was combined with the guiding peptide sequence of saccharomyces cerevisiae to form the signal peptide FLO10- αpro.FLO10 αpro is a combined signal peptide, which enhance the enzyme activity 3 times.


Characterization

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

Figure1. AOX1-FLO10-SLAC-His tag.

DNA Gel Electrophoretic

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 2208 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 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.

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.