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Team:Penn/SurfaceDisplayOverview
From 2012.igem.org
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We chose to use the Ice Nucleation Protein (Figure 1) as our surface display carrier protein. 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. | We chose to use the Ice Nucleation Protein (Figure 1) as our surface display carrier protein. 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. | ||
Revision as of 02:42, 27 October 2012
Surface Display and Targeting
*Schematic* 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:
- Achieve the first display of DARPin H10-2-G3 on the surface of E. coli and verify that the system targets cancer cells.
- 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. 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
