Difference between revisions of "Template:Team:TU Eindhoven/Experimental Approach HTML"
| Line 23: | Line 23: | ||
</span> | </span> | ||
| − | + | <br /> | |
<br /> | <br /> | ||
<hr> | <hr> | ||
Revision as of 14:02, 4 August 2015
Experimental approach
To test the viability of the designed system, we have designed a number of experiments. These experiments are conducted to verify whether the individual elements of our device work. An overview of the experiments is given below.
Verifying the click reaction
A vital aspect of our device is clicking the aptamers to the membrane proteins. For this click, we madeuse of the exact same click chemistry used by iGEM TU Eindhoven 2014. iGEM TU Eindhoven 2014 has used the click reaction N-terminally. To analyze whether the localization of the azide-functionalized amino acid within the loops of OmpX impedes the click reaction, we clicked a DBCO-functionalized fluorophore (TAMRA) to the outer membrane proteins. After some washing steps and spinning down, we expected the cells to remain fluorescent. To analyze the fluorescence at the single-cell level, we measured cells using the Fluorescence-Activated Cell Sorter (FACS). Martijn van Rosmalen gave us a clarifying FACS introduction to get us started and Wiggert Altenburg assisted us with the first experiments.
[1] American Cancer Society. Colorectal Cancer Facts & Figures 2011-2013. Atlanta: American Cancer Society, 2011.
[2] W. Zhou, P.-J. J. Huang, J. Ding, and J. Liu, “Aptamer-based biosensors for biomedical diagnostics.,” Analyst, vol. 139, no. 11, pp. 2627–40, 2014.
[3] C. Tuerk and L. Gold, “Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase.,” Science, vol. 249, no. 4968, pp. 505–10, Aug. 1990.
[4] D. Musumeci and D. Montesarchio, “Polyvalent nucleic acid aptamers and modulation of their activity: A focus on the thrombin binding aptamer,” Pharmacol. Ther., vol. 136, no. 2, pp. 202–215, 2012.
[5] A. Cibiel, C. Pestourie, and F. Ducongé, “In vivo uses of aptamers selected against cell surface biomarkers for therapy and molecular imaging,” Biochimie, vol. 94, no. 7, pp. 1595–1606, 2012.
[6] L. H. Lauridsen and R. N. Veedu, “Nucleic acid aptamers against biotoxins: a new paradigm toward the treatment and diagnostic approach.,” Nucleic Acid Ther., vol. 22, no. 6, pp. 371–9, 2012.
[7] A. Rhouati, C. Yang, A. Hayat, and J. L. Marty, “Aptamers: A promosing tool for ochratoxin a detection in food analysis,” Toxins (Basel)., vol. 5, no. 11, pp. 1988–2008, 2013.
[8] F. Radom, P. M. Jurek, M. P. Mazurek, J. Otlewski, and F. Jeleń, “Aptamers: Molecules of great potential,” Biotechnol. Adv., vol. 31, no. 8, pp. 1260–1274, 2013.
[9] B. J. Hicke, A. W. Stephens, T. Gould, Y.-F. Chang, C. K. Lynott, J. Heil, S. Borkowski, C.-S. Hilger, G. Cook, S. Warren, and P. G. Schmidt, “Tumor Targeting by an Aptamer,” J. Nucl. Med., vol. 47, no. 4, pp. 668–678, Apr. 2006.
[10] Y. Wu, K. Sefah, H. Liu, R. Wang, and W. Tan, “DNA aptamer-micelle as an efficient detection/delivery vehicle toward cancer cells.,” Proc. Natl. Acad. Sci. U. S. A., vol. 107, no. 1, pp. 5–10, 2010.
[11] A. D. Kent, N. G. Spiropulos, and J. M. Heemstra, “General approach for engineering small-molecule-binding DNA split aptamers,” Anal. Chem., vol. 85, no. 20, pp. 9916–9923, 2013.
[12] J. J. Rice, A. Schohn, P. H. Bessette, K. T. Boulware, and P. S. Daugherty, “Bacterial display using circularly permuted outer membrane protein OmpX yields high affinity peptide ligands.,” Protein Sci., vol. 15, no. 4, pp. 825–36, Apr. 2006.
[13] J. Vogt and G. E. Schulz, “The structure of the outer membrane protein OmpX from Escherichia coli reveals possible mechanisms of virulence.,” Structure, vol. 7, no. 10, pp. 1301–9, Oct. 1999.
[14] W. Alberts, Johnson, Lewis, Raff, Roberts, Molecular Biology of The Cell. Pearson, 2005.
[15] I. Medintz and N. Hildebrandt, Eds., FRET - Förster Resonance Energy Transfer. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co. KGaA, 2013.
[2] W. Zhou, P.-J. J. Huang, J. Ding, and J. Liu, “Aptamer-based biosensors for biomedical diagnostics.,” Analyst, vol. 139, no. 11, pp. 2627–40, 2014.
[3] C. Tuerk and L. Gold, “Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase.,” Science, vol. 249, no. 4968, pp. 505–10, Aug. 1990.
[4] D. Musumeci and D. Montesarchio, “Polyvalent nucleic acid aptamers and modulation of their activity: A focus on the thrombin binding aptamer,” Pharmacol. Ther., vol. 136, no. 2, pp. 202–215, 2012.
[5] A. Cibiel, C. Pestourie, and F. Ducongé, “In vivo uses of aptamers selected against cell surface biomarkers for therapy and molecular imaging,” Biochimie, vol. 94, no. 7, pp. 1595–1606, 2012.
[6] L. H. Lauridsen and R. N. Veedu, “Nucleic acid aptamers against biotoxins: a new paradigm toward the treatment and diagnostic approach.,” Nucleic Acid Ther., vol. 22, no. 6, pp. 371–9, 2012.
[7] A. Rhouati, C. Yang, A. Hayat, and J. L. Marty, “Aptamers: A promosing tool for ochratoxin a detection in food analysis,” Toxins (Basel)., vol. 5, no. 11, pp. 1988–2008, 2013.
[8] F. Radom, P. M. Jurek, M. P. Mazurek, J. Otlewski, and F. Jeleń, “Aptamers: Molecules of great potential,” Biotechnol. Adv., vol. 31, no. 8, pp. 1260–1274, 2013.
[9] B. J. Hicke, A. W. Stephens, T. Gould, Y.-F. Chang, C. K. Lynott, J. Heil, S. Borkowski, C.-S. Hilger, G. Cook, S. Warren, and P. G. Schmidt, “Tumor Targeting by an Aptamer,” J. Nucl. Med., vol. 47, no. 4, pp. 668–678, Apr. 2006.
[10] Y. Wu, K. Sefah, H. Liu, R. Wang, and W. Tan, “DNA aptamer-micelle as an efficient detection/delivery vehicle toward cancer cells.,” Proc. Natl. Acad. Sci. U. S. A., vol. 107, no. 1, pp. 5–10, 2010.
[11] A. D. Kent, N. G. Spiropulos, and J. M. Heemstra, “General approach for engineering small-molecule-binding DNA split aptamers,” Anal. Chem., vol. 85, no. 20, pp. 9916–9923, 2013.
[12] J. J. Rice, A. Schohn, P. H. Bessette, K. T. Boulware, and P. S. Daugherty, “Bacterial display using circularly permuted outer membrane protein OmpX yields high affinity peptide ligands.,” Protein Sci., vol. 15, no. 4, pp. 825–36, Apr. 2006.
[13] J. Vogt and G. E. Schulz, “The structure of the outer membrane protein OmpX from Escherichia coli reveals possible mechanisms of virulence.,” Structure, vol. 7, no. 10, pp. 1301–9, Oct. 1999.
[14] W. Alberts, Johnson, Lewis, Raff, Roberts, Molecular Biology of The Cell. Pearson, 2005.
[15] I. Medintz and N. Hildebrandt, Eds., FRET - Förster Resonance Energy Transfer. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co. KGaA, 2013.