Team:Penn/SurfaceDisplayOverview

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<b><div class="name" align="center">Objectives</div></b>
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<b><div class="name" align="center">Surface Display and Targeting</div></b><br>
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*Schematic*
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<div align="center"><img style="align:center;"src="https://static.igem.org/mediawiki/2012/a/a4/Surfacedispschematic.gif" height="525" width="700"></div><br>
While engineering bacteria as a light-activated drug delivery <a href="https://2012.igem.org/Team:Penn/LightActivatedOverview "> system </a>  is novel and useful by itself, the targeting potential of our light-activated bacterial therapeutic would be greatly improved if the bacteria also could target specific cells. The goal of the second module of our system is to allow bacterial targeting to cancer cells.  We sought to achieve this by displaying an engineered cancer cell binding protein on the surface of our <i> E. coli</i>. We successfully displayed DARPin H10-2-G3, an antibody-mimetic protein rationally evolved to picomolar affinity with HER2, a breast cancer biomarker. We verified that bacteria displaying this protein can selectively bind to breast cancer cells <i> in vitro</i>. This class of protein has not been displayed before on the surface of bacteria. Along the way, we developed a generalized BioBrick surface display system which allows future iGEM teams to display proteins of their choosing on the surface of bacteria.
While engineering bacteria as a light-activated drug delivery <a href="https://2012.igem.org/Team:Penn/LightActivatedOverview "> system </a>  is novel and useful by itself, the targeting potential of our light-activated bacterial therapeutic would be greatly improved if the bacteria also could target specific cells. The goal of the second module of our system is to allow bacterial targeting to cancer cells.  We sought to achieve this by displaying an engineered cancer cell binding protein on the surface of our <i> E. coli</i>. We successfully displayed DARPin H10-2-G3, an antibody-mimetic protein rationally evolved to picomolar affinity with HER2, a breast cancer biomarker. We verified that bacteria displaying this protein can selectively bind to breast cancer cells <i> in vitro</i>. This class of protein has not been displayed before on the surface of bacteria. Along the way, we developed a generalized BioBrick surface display system which allows future iGEM teams to display proteins of their choosing on the surface of bacteria.
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<b><div class="name" align="center">Ice Nucleation Protein</div></b>
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The <a href="http://www.bioc.uzh.ch/plueckthun/"> lab </a>   of Andreas Plueckthun at ETH Zurich has pioneered the development of Designed Ankyrin Repeat Protein (DARPin) technology.  DARPins are engineered antibody-mimetic proteins consisting of 3-5 ankyrin repeat motifs. These proteins have been rationally evolved to high binding affinity with targets through ribosome display. In 2007, the group developed H10-2-G3 [1], a  14.7kDa DARPin  evolved to 90pM affinity with the extracellular domain of HER2 (Figure 1).
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We chose to use the Ice Nucleation Protein (Figure 1) as our surface display carrier protein. Ice nucleation protein (INP) is a protein found in <i>Xanthomonas campestris</i> pc. campestris BCRC 12846. Its function is to provide a surface for ice nucleation, which results in the formation of ice crystals. However, recent studies have utilized INP for its surface display properties. In nature, the protein is anchored in the membrane through a glycosylphosphatidylinositol (GPI) anchor, a relatively rare occurrence in prokaryotes. The Ice Nucleation Protein protein has been used to display enzymes [1], typically for biocatalysis applications (e.g. Edinburgh iGEM 2011). To make the protein a more manageable size, we truncated the protein to the N and C terminal domains only. The C terminal domain is displayed at the cell surface, while the N terminal domain remains in the outer membrane. We sought to apply this system to health/medicine by displaying DARPin H10-2-G3.
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<div class="fig11"><b>Figure 1</b>: Structure of DARPin-H10-2-G3.</p></div>
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<div class="fig"><div align="center"><img src="https://static.igem.org/mediawiki/2012/e/e4/INPNC.png" /><br>
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<b>Figure 1</b></div><div style="text-align:center">Figure 1: Proposed Structure of Ice Nucleation Protein.</div></div>
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[1] Zahnd, C., Wyler, E., Schwenk, J. M., Steiner, D., Lawrence, M. C., McKern, N. M., Pecorari, F., et al. (2007). A designed ankyrin repeat protein evolved to picomolar affinity to Her2. Journal of molecular biology, 369(4), 1015–28. doi:10.1016/j.jmb.2007.03.028
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[1] Zahnd, C., Wyler, E., Schwenk, J. M., Steiner, D., Lawrence, M. C., McKern, N. M., Pecorari, F., et al. (2007). A designed ankyrin repeat protein evolved to picomolar affinity to Her2. Journal of molecular biology, 369(4), 1015–28. doi:10.1016/j.jmb.2007.03.028
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<b><div class="name" align="center">DARPin H10-2-G3</div></b>
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The <a href="http://www.bioc.uzh.ch/plueckthun/"> lab </a>  of Andreas Plueckthun at ETH Zurich has pioneered the development of Designed Ankyrin Repeat Protein (DARPin) technology. DARPins are engineered antibody-mimetic proteins consisting of 3-5 ankyrin repeat motifs. These proteins have been rationally evolved to high binding affinity with targets through ribosome display. In 2007, the group developed H10-2-G3 [2], a  14.7kDa DARPin  evolved to 90pM affinity with the extracellular domain of HER2 (Figure 2).
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<div class="fig"><div align="center"><img src="https://static.igem.org/mediawiki/2012/b/b7/DARPingif.gif" /><br>
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<b>Figure 2</b></div><div style="text-align:center">Figure 2: Structure of DARPin-H10-2-G3.</div></div>
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[2] Zahnd, C., Wyler, E., Schwenk, J. M., Steiner, D., Lawrence, M. C., McKern, N. M., Pecorari, F., et al. (2007). A designed ankyrin repeat protein evolved to picomolar affinity to Her2. Journal of molecular biology, 369(4), 1015–28. doi:10.1016/j.jmb.2007.03.028  
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Latest revision as of 03:48, 27 October 2012

Penn 2012 iGEM Wiki

Image Map

Surface Display and Targeting


While engineering bacteria as a light-activated drug delivery system is novel and useful by itself, the targeting potential of our light-activated bacterial therapeutic would be greatly improved if the bacteria also could target specific cells. The goal of the second module of our system is to allow bacterial targeting to cancer cells. We sought to achieve this by displaying an engineered cancer cell binding protein on the surface of our E. coli. We successfully displayed DARPin H10-2-G3, an antibody-mimetic protein rationally evolved to picomolar affinity with HER2, a breast cancer biomarker. We verified that bacteria displaying this protein can selectively bind to breast cancer cells in vitro. This class of protein has not been displayed before on the surface of bacteria. Along the way, we developed a generalized BioBrick surface display system which allows future iGEM teams to display proteins of their choosing on the surface of bacteria.
Objectives

Our goals were twofold:

  1. Achieve the first display of DARPin H10-2-G3 on the surface of E. coli and verify that the system targets cancer cells.
  2. Create a generalized BioBrick surface display platform for other labs and iGEM teams.
Ice Nucleation Protein
We chose to use the Ice Nucleation Protein (Figure 1) as our surface display carrier protein. Ice nucleation protein (INP) is a protein found in Xanthomonas campestris pc. campestris BCRC 12846. Its function is to provide a surface for ice nucleation, which results in the formation of ice crystals. However, recent studies have utilized INP for its surface display properties. In nature, the protein is anchored in the membrane through a glycosylphosphatidylinositol (GPI) anchor, a relatively rare occurrence in prokaryotes. The Ice Nucleation Protein protein has been used to display enzymes [1], typically for biocatalysis applications (e.g. Edinburgh iGEM 2011). To make the protein a more manageable size, we truncated the protein to the N and C terminal domains only. The C terminal domain is displayed at the cell surface, while the N terminal domain remains in the outer membrane. We sought to apply this system to health/medicine by displaying DARPin H10-2-G3.

Figure 1
Figure 1: Proposed Structure of Ice Nucleation Protein.



[1] Zahnd, C., Wyler, E., Schwenk, J. M., Steiner, D., Lawrence, M. C., McKern, N. M., Pecorari, F., et al. (2007). A designed ankyrin repeat protein evolved to picomolar affinity to Her2. Journal of molecular biology, 369(4), 1015–28. doi:10.1016/j.jmb.2007.03.028


DARPin H10-2-G3
The lab of Andreas Plueckthun at ETH Zurich has pioneered the development of Designed Ankyrin Repeat Protein (DARPin) technology. DARPins are engineered antibody-mimetic proteins consisting of 3-5 ankyrin repeat motifs. These proteins have been rationally evolved to high binding affinity with targets through ribosome display. In 2007, the group developed H10-2-G3 [2], a 14.7kDa DARPin evolved to 90pM affinity with the extracellular domain of HER2 (Figure 2).

Figure 2
Figure 2: Structure of DARPin-H10-2-G3.



[2] Zahnd, C., Wyler, E., Schwenk, J. M., Steiner, D., Lawrence, M. C., McKern, N. M., Pecorari, F., et al. (2007). A designed ankyrin repeat protein evolved to picomolar affinity to Her2. Journal of molecular biology, 369(4), 1015–28. doi:10.1016/j.jmb.2007.03.028


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