Difference between revisions of "Part:BBa K3196011"
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K3196011 short</partinfo>
 
<partinfo>BBa_K3196011 short</partinfo>
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FLO10 is a Lectin-like protein with similarity to Flo1 p, thought to be expressed and involved in flocculation. SLAC can catalyze lignin.
 
  
 
<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 composite part that used to degraded lignin.
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[[File:T--HUST--China--2019-FLO10SLAC.jpg ‎|525px|thumb|center|Figure1. AOX1-FLO10-SLAC-His tag.]]
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SLAC is a multicopper oxidase isolated from S. coelicolor , capable of catalyzing one-electron oxidation of a wide range of substrates to generate radicals while concomitantly reducing molecular oxygen to water<sup>[1]</sup>.
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 +
FLO10 is signal peptide suitable for expression in  pichia pastoris
 +
Lectin-like protein with similarity to Flo1 p，thought to be expressed andinvolved in flocculation.<sup>[2]</sup>
 +
 
 +
P. pastoris is usually the preferred host for the production of industrial enzymes.
 +
 
 +
[[File:T--HUST--China--2019-FLO10-αproSLAC.jpg ‎|400px|thumb|center|Figure1.This is the pathway of this composite part.]]
  
 
<h1>'''DNA Gel Electrophoretic'''</h1>
 
<h1>'''DNA Gel Electrophoretic'''</h1>
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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 2009 bp which means the PCR is successful.
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To confirm the function of this part, first we confirm that the gene is transferred to P. pastoris GS115 successfully.
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[[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. ]]
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1.DNA extraction of the E.coli plasmid and verification of the right fragment.
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2.Prepare the competent cells of P. pastoris GS115.
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3.Electro transformation.
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4.Yeast genome extraction and PCR verification.
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As the picture shows, we have constructed the engineering bacteria successfully.
 +
[[File:T--HUST-China--2019-FLO10-SLAC-gel.png |400px|thumb|center|Figure1:This is the DNA Gel Electrophoretic after the PCR of engineering P. pastoris GS115 genomo. ]]
 +
 
  
 
<h1>'''SDS-PAGE'''</h1>
 
<h1>'''SDS-PAGE'''</h1>
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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.
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Second, we cultured the engineering P. pastoris GS115(FLO10-SLAC)in the buffered glycerol-complex medium (BMGY) and induced it in buffered minimal methanol medium (BMM).  
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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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[[File:T--HUST-China--2019-SLAC-SDS-PAGE.jpg |400px|thumb|center|Figure3:The SDS-page result shows that engineering P. pastoris GS115(FLO10-SLAC) have successfully secrete SLAC protein into superfluous liquid.]]
  
  
 
<h1>'''Enzyme Activity'''</h1>
 
<h1>'''Enzyme Activity'''</h1>
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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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Laccase activity was determined at room temperature (22–25 °C) using ABTS. Oxidation of ABTS (1 mM) was measured at 420 nm (ε = 36,000 M−1 cm−1) in 20 mM acetate buffer (pH 4.0).  
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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, PHO1apro shows a more delayed increase on enzyme activity.]]
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 +
By using this formula:
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〖activity=(A2−A1)〗∕t∗11244
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 +
We obtain the follow figure that represent the enzyme activity changes with time.  
 +
 
 +
[[File:T--HUST-China--2019-FLO10-SLAC.png |400px|thumb|center|Figure4:This is the engineering P. pastoris GS115(FLO10-SLAC) enzyme activity curve. ]]
  
 
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Revision as of 12:11, 21 October 2019


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

Characterization

This is a composite part that used to degraded lignin. SLAC is a multicopper oxidase isolated from S. coelicolor , capable of catalyzing one-electron oxidation of a wide range of substrates to generate radicals while concomitantly reducing molecular oxygen to water[1].

FLO10 is signal peptide suitable for expression in pichia pastoris Lectin-like protein with similarity to Flo1 p，thought to be expressed andinvolved in flocculation.[2]

P. pastoris is usually the preferred host for the production of industrial enzymes.

Figure1.This is the pathway of this composite part.

DNA Gel Electrophoretic

To confirm the function of this part, first we confirm that the gene is transferred to P. pastoris GS115 successfully.

1.DNA extraction of the E.coli plasmid and verification of the right fragment.

2.Prepare the competent cells of P. pastoris GS115.

3.Electro transformation.

4.Yeast genome extraction and PCR verification.

As the picture shows, we have constructed the engineering bacteria successfully.

Figure1:This is the DNA Gel Electrophoretic after the PCR of engineering P. pastoris GS115 genomo.


SDS-PAGE

Second, we cultured the engineering P. pastoris GS115(FLO10-SLAC)in the buffered glycerol-complex medium (BMGY) and induced it in buffered minimal methanol medium (BMM).

Figure3:The SDS-page result shows that engineering P. pastoris GS115(FLO10-SLAC) have successfully secrete SLAC protein into superfluous liquid.


Enzyme Activity

Laccase activity was determined at room temperature (22–25 °C) using ABTS. Oxidation of ABTS (1 mM) was measured at 420 nm (ε = 36,000 M−1 cm−1) in 20 mM acetate buffer (pH 4.0).

By using this formula: 〖activity=(A2−A1)〗∕t∗11244

We obtain the follow figure that represent the enzyme activity changes with time.

Figure4:This is the engineering P. pastoris GS115(FLO10-SLAC) enzyme activity curve.

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
    Illegal BamHI site found at 948
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1387