Difference between revisions of "Part:BBa K1921007"

 
 
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<partinfo>BBa_K1921007 parameters</partinfo>
 
<partinfo>BBa_K1921007 parameters</partinfo>
 
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 +
===Usage===
 +
As a cell wall protein of Pichia pastoris, GCW61 is often used as an anchor protein in Pichia pastoris surface display system because of its stable connection with the dextranomer in cell wall.  By fusing GCW61 with the target protein expression, the target protein can be displayed on the outer of the yeast cell wall. <br>
 +
By expressing the fusion protein of GCW61 protein and PETase, PETase was expressed on the surface of Pichia pastoris, and the whole cell catalyst for the degradation of PET was obtained. We made the PETase fixed on the cell wall, which can improve the stability of PETase, and is easy to control the degradation reaction of PET and PETase recycling.<br>
 +
We displayed the hydrophobic protein inJanus/sJanus on Pichia pastoris cells by the fusion expression of GCW61 and the hydrophobic protein inJanus/sJanus. Due to the nature that hydrophobic protein can independently mounted on any interface to form a layer of amphipathic membrane to reverse the hydrophilic - hydrophobic property of the interface, the hydrophobic protein displayed on the cell surface can greatly enhance the hydrophobic surface of yeast cells, provide the hydrophobic environment required for the degradation reaction of PET by PETase.<br>
 +
 +
===Biology===
 +
The part was gained from Pichia pastoris GS115.<br>
 +
As one of the Glycosylphosphatidylinositoled cell wall proteins (GPI-CWPs), GCW61 is located in the outer layer of yeast cell wall, its C terminal is oligo mannose glycosylated. Subsequently, the mannose chain of GCW61 connect with the β-1,6 dextranomer of inner cell wall layer by forming covalent connection, thus, the GCW61 is fixed in the outer layer of the cell wall protein.<br>
 +
 +
===Reference===
 +
[1] Kinoshita T, Fujiata M. Overview of GPI biosynthesis [J]. The enzymes. 2009;26:1-30. <br>
 +
[2] Orlean P, Mennon AK. Thematic review series: lipid posttranslational modifications. GPI anchoring of protein in yeast and mammalian cells, or: how we learned to stop worrying and love glycophospholipids [J]. Journal of lipid research.2007;48(5):993-1011. <br>
 +
[3] Mouyna I, Fontaine T, Vai M, et al. Glycosylphosphatidy linositol-anchored glucanosyltransferases play an active role in the biosynthesis of the fungal cell wall[J]. Journal of Biological Chemistry.2000;275(20):14882-14889.
 +
 +
===Surface display HPLC Results===
 +
'''Surface display in Pichia Pastoris:'''<br>
 +
<p style="text-align: center;">
 +
    https://static.igem.org/mediawiki/igem.org/d/d7/ProofTJU15.jpg<br>
 +
</p>
 +
<p style="text-align: center;">
 +
    https://static.igem.org/mediawiki/igem.org/0/0c/ProofTJU16.jpg<br>
 +
'''Figure 1.''' The activity of P. pastoris PETase-GCW61. a&b used the first group of yeast; c&d used the third of yeast; a&c:the activity in different yeasts'concentration under the best hour; b&d: the activity in different hours under the best concentration.<br>
 +
</p>
 +
 +
'''Co-display in Pichia Pastoris:'''<br>
 +
<p style="text-align: center;">
 +
    https://static.igem.org/mediawiki/igem.org/6/67/ProofTJU17.jpg<br>
 +
'''Figure 2.''' The activity of the first group of ppic9-PETase-GCW51 & ppiczaA-sJanus-GCW61 co-display transformants in different hours  and amount of yeast.<br>
 +
</p>
 +
<p style="text-align: center;">
 +
    https://static.igem.org/mediawiki/igem.org/d/d5/ProofTJU18.jpg<br>
 +
'''Figure 3.''' The activity of the second group of ppic9-PETase-GCW51 & ppiczaA-sJanus-GCW61 co-display transformants in different hours  and amount of yeast.<br>
 +
</p>
 +
<p style="text-align: center;">
 +
    https://static.igem.org/mediawiki/igem.org/b/b9/ProofTJU19.jpg<br>
 +
'''Figure 4.''' The activity of the first group of ppic9-PETase-GCW51 & ppiczaA-inJanus-GCW61 co-display transformants in different hours  and amount of yeast.<br>
 +
</p>
 +
<p style="text-align: center;">
 +
    https://static.igem.org/mediawiki/igem.org/a/a0/ProofTJU20.jpg<br>
 +
'''Figure 5.''' The activity of the second group of ppic9-PETase-GCW51 & ppiczaA-inJanus-GCW61 co-display transformants in different hours  and amount of yeast.<br>
 +
</p>
 +
<p style="text-align: center;">
 +
    https://static.igem.org/mediawiki/igem.org/e/ea/ProofTJU21.jpg<br>
 +
'''Figure 6.''' The activity of ppic9-PETase-GCW51 & ppiczaA-inJanus-GCW61 co-display transformant and ppic9-PETase-GCW51 & ppiczaA-sJanus-GCW61 co-display transformant in best condition.<br>
 +
</p>

Latest revision as of 07:42, 20 October 2016


GCW61

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage

As a cell wall protein of Pichia pastoris, GCW61 is often used as an anchor protein in Pichia pastoris surface display system because of its stable connection with the dextranomer in cell wall.  By fusing GCW61 with the target protein expression, the target protein can be displayed on the outer of the yeast cell wall. 
By expressing the fusion protein of GCW61 protein and PETase, PETase was expressed on the surface of Pichia pastoris, and the whole cell catalyst for the degradation of PET was obtained. We made the PETase fixed on the cell wall, which can improve the stability of PETase, and is easy to control the degradation reaction of PET and PETase recycling.
We displayed the hydrophobic protein inJanus/sJanus on Pichia pastoris cells by the fusion expression of GCW61 and the hydrophobic protein inJanus/sJanus. Due to the nature that hydrophobic protein can independently mounted on any interface to form a layer of amphipathic membrane to reverse the hydrophilic - hydrophobic property of the interface, the hydrophobic protein displayed on the cell surface can greatly enhance the hydrophobic surface of yeast cells, provide the hydrophobic environment required for the degradation reaction of PET by PETase.

Biology

The part was gained from Pichia pastoris GS115.
As one of the Glycosylphosphatidylinositoled cell wall proteins (GPI-CWPs), GCW61 is located in the outer layer of yeast cell wall, its C terminal is oligo mannose glycosylated. Subsequently, the mannose chain of GCW61 connect with the β-1,6 dextranomer of inner cell wall layer by forming covalent connection, thus, the GCW61 is fixed in the outer layer of the cell wall protein.

Reference

[1] Kinoshita T, Fujiata M. Overview of GPI biosynthesis [J]. The enzymes. 2009;26:1-30.
[2] Orlean P, Mennon AK. Thematic review series: lipid posttranslational modifications. GPI anchoring of protein in yeast and mammalian cells, or: how we learned to stop worrying and love glycophospholipids [J]. Journal of lipid research.2007;48(5):993-1011.
[3] Mouyna I, Fontaine T, Vai M, et al. Glycosylphosphatidy linositol-anchored glucanosyltransferases play an active role in the biosynthesis of the fungal cell wall[J]. Journal of Biological Chemistry.2000;275(20):14882-14889.

Surface display HPLC Results

Surface display in Pichia Pastoris:

ProofTJU15.jpg

ProofTJU16.jpg
Figure 1. The activity of P. pastoris PETase-GCW61. a&b used the first group of yeast; c&d used the third of yeast; a&c:the activity in different yeasts'concentration under the best hour; b&d: the activity in different hours under the best concentration.

Co-display in Pichia Pastoris:

ProofTJU17.jpg
Figure 2. The activity of the first group of ppic9-PETase-GCW51 & ppiczaA-sJanus-GCW61 co-display transformants in different hours and amount of yeast.

ProofTJU18.jpg
Figure 3. The activity of the second group of ppic9-PETase-GCW51 & ppiczaA-sJanus-GCW61 co-display transformants in different hours and amount of yeast.

ProofTJU19.jpg
Figure 4. The activity of the first group of ppic9-PETase-GCW51 & ppiczaA-inJanus-GCW61 co-display transformants in different hours and amount of yeast.

ProofTJU20.jpg
Figure 5. The activity of the second group of ppic9-PETase-GCW51 & ppiczaA-inJanus-GCW61 co-display transformants in different hours and amount of yeast.

ProofTJU21.jpg
Figure 6. The activity of ppic9-PETase-GCW51 & ppiczaA-inJanus-GCW61 co-display transformant and ppic9-PETase-GCW51 & ppiczaA-sJanus-GCW61 co-display transformant in best condition.