Difference between revisions of "Part:BBa K3196017"
 
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<h1>'''Characterization'''</h1>
 
<h1>'''Characterization'''</h1>
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This is a four section for degrade and transfer lignin part.
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This is a four section for degrade and transfer pectin part.
 
[[File:T--HUST--China--2019-FLO10-αpropelA.jpg ‎|400px|thumb|center|Figure1. T--HUST--China--2019-FLO10-αpropelA]]
 
[[File:T--HUST--China--2019-FLO10-αpropelA.jpg ‎|400px|thumb|center|Figure1. T--HUST--China--2019-FLO10-αpropelA]]
  
 
<h1>'''DNA Gel Electrophoretic'''</h1>
 
<h1>'''DNA Gel Electrophoretic'''</h1>
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After we link FLO10 αpro and pelA successfully, we run the PCR with an intention to confirm the expression of pelA. As the figure shows, we get the genetic stripe about xxx bp which means the PCR is successful.
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After we link FLO10 αpro and pelA successfully, we run the PCR with an intention to confirm the expression of pelA. As the figure shows, we get the genetic stripe about 1092 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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Electro-transformation to Pichia pastoris was all succeeded.
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[[File:T--HUST--China--2019-pelA_Gel.png|400px|thumb|center| Figure1 Yeast Genome extraction and pcr]]
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<h1>'''SDS-PAGE'''</h1>
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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.
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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>'''Enzyme Activity'''</h1>
 
<h1>'''Enzyme Activity'''</h1>
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We use DNS to detect the enzyme activity. As the figure shows, the solution turns xxx, which confirm the enzyme activity.
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The enzyme activity of the pelA group is obtained by titration and daily measurement of 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-pelA data.jpg |400px|thumb|center| Figure2 Determination of pelA enzyme activity]]
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Pectin is a hydrophilic polysaccharide substance present in the interstitial of plant cells. It is stable in acidic medium and insoluble in ethanol. It is a natural polymer compound. We understand that pectinase measures enzyme activity has a nationally standardized experimental procedure, so we have adopted the standard titration scheme given by the state. <sup> [3] </sup>
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Latest revision as of 11:34, 20 October 2019


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

FLO10 αpro is a combined signal peptide, which enhance the enzyme activity 3 times. pelA can catalyze pectin.

Characterization

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

Figure1. T--HUST--China--2019-FLO10-αpropelA

DNA Gel Electrophoretic

After we link FLO10 αpro and pelA successfully, we run the PCR with an intention to confirm the expression of pelA. As the figure shows, we get the genetic stripe about 1092 bp which means the PCR is successful. Electro-transformation to Pichia pastoris was all succeeded.

Figure1 Yeast Genome extraction and pcr

Enzyme Activity

The enzyme activity of the pelA group is obtained by titration and daily measurement of enzyme activity.

Figure2 Determination of pelA enzyme activity


Pectin is a hydrophilic polysaccharide substance present in the interstitial of plant cells. It is stable in acidic medium and insoluble in ethanol. It is a natural polymer compound. We understand that pectinase measures enzyme activity has a nationally standardized experimental procedure, so we have adopted the standard titration scheme given by the state. [3]


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 1986
    Illegal BamHI site found at 937
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]