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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===Usage and Biology===
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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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<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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<regulartext1> <b><br><BR><BR><BR><BR><br><BR><BR><BR><BR><bR><BR><BR><BR><BR>Figure 2:</b> Experiment, induced mgfp-
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5 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></html>
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Revision as of 03:31, 29 September 2011

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 mgfp- 5 yeast. 3-D cluster of yeast cells growing around and on top of other cells.