Template:Team:TU Eindhoven/Experimental Approach HTML
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)
.
A Fluorescence-Activated Cell Sorter (FACS) is a specialized flow cytometer (see Figure X). The presence of a cell and cell size is detected using light scattering. This information is combined with the fluorescent characteristics of each cell. Based on these properties, the cells can be sorted. iGEM TU Eindhoven 2014 has written an extensive piece on the FACS which can be found here.
Martijn van Rosmalen is a PhD student at Eindhoven University within the Merkx Group. In his research, he aims to apply yeast display and fluorescence activated cell sorting to the development of new FRET sensors. Martijn van Rosmalen maintains the FACS and took the time to familiarize us with the FACS.
gave us a clarifying FACS introduction to get us started and Wiggert Altenburg
Wiggert Altenburg is currently a first-years master student. Wiggert was a member of iGEM TU Eindhoven 2014 and in his role he did all the experiments with the FACS. In his bachelor thesis, Wiggert carried out and finetuned the Click Chemistry experiments which were presented at 2014's Giant Jamboree as Click Coli by iGEM TU Eindhoven.
assisted us with the first experiments.
Measuring bioluminiscence & fluorescence
DNA Strand displacement
As we have already touched upon lightly, it has long been thought that nucleic acids had only a single role: carrying hereditary information. As discussed, the discovery that DNA could fold into higher-order structures gave way to SELEX, an evolutionary method of discovering aptamers. The construction of DNA nanostructures, however, was not only carried out through a combinatorial approach: the specificity and predictability of Watson-Crick basepairing enabled rational design for engineering at the nanoscale [1]. Initially, the designed DNA nanostructures were mostly static, but dynamic nanostructures have become available over the years. Most of these dynamic nanostructures share a common feature: they exploit a phenomenon known as DNA strand-displacement.
DNA Strand displacement is the workhorse of dynamic DNA technology, the field which uses DNA’s non-covalent interactions to assemble higher-order structures. DNA strand displacement is a process where two strands with partial complementarity are hybridized. These pre-assembled DNA strands have only partial complementary, leaving room for a toehold region. When a DNA sequence with full complementary binds to this region (the input), branch migration takes place (see the figure below). In the end, the sequence with full complementary binds to the sequence, yielding the output. 
DNA strand-displacement is a very robust technology: the sequences of the used strands often go unreported as they play a minor role. The robustness of the technology enabled rational design of numerous nanostructures. These nanostructures include DNA walkers, strand displacement cascades and self-assembling dendrimers [1].
In our project, we want to bring two membrane proteins in close proximity through aptamers. To test whether a close proximity yields a signal, we will use DNA to bring the membrane proteins in close proximity, as DNA’s high predictability and specificity makes it the ideal probe to bring the membrane proteins into close proximity. To come to a reversible system, we will use abovementioned strand displacement (see Figure 2).
[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.