Difference between revisions of "Part:BBa K644001"
 
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We cloned HWP1 into the plasmid pCTCON2. We used homologous recombination and transformed it into EBY100 Yeast. We used pCTCON2 because it has Aga2, which helps express HWP1 on the surface of the yeast cell. pCTCON2 also has the pGal1 promoter so HWP1 is only expressed in the presence of Galactose. A TRP marker is used as a selectable marker to tell the difference between yeast cells with the pCTCON2 plasmid and yeast cells without the pCTCON2 plasmid. ''S. cerevisiae'' will express HWP1 in the presence of Galactose and will not when it isn't present.
 
We cloned HWP1 into the plasmid pCTCON2. We used homologous recombination and transformed it into EBY100 Yeast. We used pCTCON2 because it has Aga2, which helps express HWP1 on the surface of the yeast cell. pCTCON2 also has the pGal1 promoter so HWP1 is only expressed in the presence of Galactose. A TRP marker is used as a selectable marker to tell the difference between yeast cells with the pCTCON2 plasmid and yeast cells without the pCTCON2 plasmid. ''S. cerevisiae'' will express HWP1 in the presence of Galactose and will not when it isn't present.
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<partinfo>BBa_K644001 parameters</partsinfo>
 
<partinfo>BBa_K644001 parameters</partsinfo>
 
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===Usage and Biology===
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<html>
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<regulartext> Using the pCTCON2 vector, we made a fusion protein of Hwp1 and Aga2, such that Hwp1 would be expressed and displayed on the surface of <i>S. cerevisiae</i>. pCTCON2 has a gal promoter such that the expression of Aga2-Hwp1 is controlled by the presence of galactose in the media. As shown below, only cells grown in the presence of galactose expressed Hwp1 and were able to adhere to one another in large clumps.
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<div">
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<img align="left" style="margin-bottom:0px; width: 300px; margin-top:0px; padding:0;" src="http://dl.dropbox.com/u/24404809/Hwp1ctrl.jpg">
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<regulartext1> <b><br><BR><BR><BR><BR><br><BR><BR><BR><BR><BR><bR><BR><BR><BR><BR>Figure 1:</b> Control, non-induced Hwp1 expressing yeast. Dispersed, no major
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clustering. </regulartext1>
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</div>
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<div>
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<img align="left" style="margin-bottom:0px; width: 300px; margin-top:0px; padding:0;" src="http://dl.dropbox.com/u/24404809/Hwp1Gal1.jpg">
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<BR>
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<regulartext1> <b><br><BR><BR><BR><BR><br><BR><BR><BR><BR><bR><BR><BR><BR><BR>Figure 2:</b> Experiment, induced Hwp1
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expressing yeast. 3-D cluster of yeast cells
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growing around and on top of other
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cells. </regulartext1> </div>
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</html>
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===<h1>2020 XHD-Wuhan-China’s Contribution</h1>===
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<h2> Introduction </h2>
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In this paper, the author's research shows that mucoadhesive nanogel containing farnesol has obvious antifungal activity for Candida albicans. Then authors investigated the mechanism by which farnesol reduces the infection rate of bacteria, which involves the expression of HWP1 gene.
 +
 +
<h2>Results </h2>
 +
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The effect of farnesol, CS and AL nanogels containing farnesol
 +
on the expression of HWP1, SAP6 and Rim101 genes was
 +
investigated using real-time PCR (Figure 7). The finding shows
 +
that expression of HWP1 and SAP6 genes in C. albicans treated
 +
with 300 mM concentration of farnesol, CS and AL nanogels
 +
containing farnesol decreased significantly in comparison with
 +
un-treated control group (p<.01). However, it was found the
 +
significant difference between the expression of SAP6 gene of
 +
C. albicans treated with CS nanogel and non-treated Candida
 +
was observed. The expression of Rim101 in Candida treated
 +
with CS nanogel significantly decreased as compared to the
 +
non-treated cells (p<.01), whereas, Rim101 C. albicans treated
 +
with farnesol and AL nanogel containing farnesol did not
 +
show any change in the gene expression level.
 +
 +
<li style="display: inline-block;"> [[File: T--XHD-Wuhan-China--literature_figure_2.jpeg |thumb|none|800px]]
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<h2>References</h2>
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Fatemeh Nikoomanesh, et al.Design and synthesis of mucoadhesive nanogel containing farnesol: investigation of the effect on HWP1, SAP6 and Rim101 genes expression of Candida albicans in vitro. ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 2019, VOL. 47, NO. 1, 64–72

Latest revision as of 01:06, 28 October 2020

Candida albicans hyphal wall protein 1 (HWP1) gene

HWP1 is a Candida albicans cell surface protein gene. It is used in Candida albicans to mediate tight binding to oral epithelial cells. Hwp1 is covalently linked to cell wall glucan through a remnant of its GPI anchor. (Eukaryot Cell. 2006 October; 5(10): 1604–1610.) Hwp1 is known to cause biofilm formation in vivo in C. albicans.

We cloned HWP1 into the plasmid pCTCON2. We used homologous recombination and transformed it into EBY100 Yeast. We used pCTCON2 because it has Aga2, which helps express HWP1 on the surface of the yeast cell. pCTCON2 also has the pGal1 promoter so HWP1 is only expressed in the presence of Galactose. A TRP marker is used as a selectable marker to tell the difference between yeast cells with the pCTCON2 plasmid and yeast cells without the pCTCON2 plasmid. S. cerevisiae will express HWP1 in the presence of Galactose and will not when it isn't present.


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 617
    Illegal BamHI site found at 1
    Illegal BamHI site found at 1053
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

Using the pCTCON2 vector, we made a fusion protein of Hwp1 and Aga2, such that Hwp1 would be expressed and displayed on the surface of S. cerevisiae. pCTCON2 has a gal promoter such that the expression of Aga2-Hwp1 is controlled by the presence of galactose in the media. As shown below, only cells grown in the presence of galactose expressed Hwp1 and were able to adhere to one another in large clumps.















Figure 1: Control, non-induced Hwp1 expressing yeast. Dispersed, no major clustering.

















Figure 2: Experiment, induced Hwp1 expressing yeast. 3-D cluster of yeast cells growing around and on top of other cells.

2020 XHD-Wuhan-China’s Contribution

Introduction

In this paper, the author's research shows that mucoadhesive nanogel containing farnesol has obvious antifungal activity for Candida albicans. Then authors investigated the mechanism by which farnesol reduces the infection rate of bacteria, which involves the expression of HWP1 gene.

Results

The effect of farnesol, CS and AL nanogels containing farnesol on the expression of HWP1, SAP6 and Rim101 genes was investigated using real-time PCR (Figure 7). The finding shows that expression of HWP1 and SAP6 genes in C. albicans treated with 300 mM concentration of farnesol, CS and AL nanogels containing farnesol decreased significantly in comparison with un-treated control group (p<.01). However, it was found the significant difference between the expression of SAP6 gene of C. albicans treated with CS nanogel and non-treated Candida was observed. The expression of Rim101 in Candida treated with CS nanogel significantly decreased as compared to the non-treated cells (p<.01), whereas, Rim101 C. albicans treated with farnesol and AL nanogel containing farnesol did not show any change in the gene expression level.

  • T--XHD-Wuhan-China--literature figure 2.jpeg

    References

    Fatemeh Nikoomanesh, et al.Design and synthesis of mucoadhesive nanogel containing farnesol: investigation of the effect on HWP1, SAP6 and Rim101 genes expression of Candida albicans in vitro. ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 2019, VOL. 47, NO. 1, 64–72