Difference between revisions of "Part:BBa K3196020"
 
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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-SUC2pelA.jpg ‎|400px|thumb|center|Figure1. T--HUST--China--2019-SUC2pelA]]
 
[[File:T--HUST--China--2019-SUC2pelA.jpg ‎|400px|thumb|center|Figure1. T--HUST--China--2019-SUC2pelA]]
  
 
<h1>'''DNA Gel Electrophoretic'''</h1>
 
<h1>'''DNA Gel Electrophoretic'''</h1>
−
After we link SUC2 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.
+
After we link SUC2 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.
 +
[[File:T--HUST--China--2019-pelA_Gel.png|400px|thumb|center| Figure1 Yeast Genome extraction and pcr]]
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<h1>'''Enzyme Activity'''</h1>
 +
The enzyme activity of the pelA group is obtained by titration and daily measurement of enzyme activity.
 +
[[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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<h1>'''SDS-PAGE'''</h1>
 
−
We run the SDS-PAGE to check whether SUC2 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.]]
 
  
  

Latest revision as of 11:37, 20 October 2019


AOX1-Kozak-SUC2-pelA-His tag-AOX1 Terminator

The SUC2 gene of S. cerevisiae, encoding invertase was designed to codon preference of P. pastoris excluding the sequence of the N-terminal pre-pro-sequence signal peptide and His-tag in the C terminus. pelA can catalyze pectin.

Characterization

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

Figure1. T--HUST--China--2019-SUC2pelA

DNA Gel Electrophoretic

After we link SUC2 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 1761
    Illegal BamHI site found at 937
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]