Team:Freiburg/Project

From 2008.igem.org

(Difference between revisions)

Revision as of 21:19, 29 October 2008

Project Report

_project report

Summary

Modular Synthetic Receptor System

In the following we provide a summary of our project and display the highlights of our achievements. For a more detailed view please see the reports of each sub-project.
The general goal of the Freiburg 2008 team is to establish a synthetic transmembrane receptor system comprising the extracellular input devices, the extracellular receiver domain, the transducer to the cytosol and the reporter/executor in the cytosol.
As controllable receptor activation event, we chose spatial control of dimerization, which is a mechanism often used by nature. To achieve spatial control in the nanometer range of proteins we chose DNA origami for exact and complex patterns with several ligands and chemical coupling of one type of ligand to scaffold proteins for simple multimerization.
To facilitate binding, we used a single-chain Fv fragment and a designed anticalin. Both bind haptens which can be conjugated to the DNA-Origami or scaffold proteins. Transduction to the cytosole is mediated by a single transmembrane helix.
As intracellular reporters we employ split proteins. Here, we used the split lactamase from the iGEM2007 Freiburg project as well as the split fluorescent proteins CFP/cerulean and YFP/venus. To provide steric flexibility and reach of our binding and reporting modules we interspersed linkers between each functional domain.
All parts are cloned in in E. coli and feature full BioBrick-compatibility. However, we test our receptors in human or mouse cells/chassis. Thus, to direct our constructs to the cell surface we attached a eukaryotic signal peptide to the N-terminus of each synthetic receptor. In addition to the new synthetic receptors, we also employed an existing T-cell line displaying a TCR fused to a single chain as a test for the spatial control.
Besides designing and cloning parts we spent quite some time on establishing and analyzing the biological experiment. In particular we wanted to test the spatial control via DNA origami using an existing cell line expressing the scFv fused to a T-cell receptor. Here we addressed the following questions:

- Can we improve DNA origami assembly yield by varying the staple oligonucleotide to scaffold DNA ratio?
- Can we find buffer conditions, which mediate cell viability and origami stability?
-Can we downsize a Ca2+ release assay to fit the sample sizes of our precious DNA origami?
- Do our constructs express and then localize in the membrane of eukaryotic cells?
-Can we detect synthetic receptor activation?

To reach our goal within the short given time frame we started several subprojects in parallel. Our sub-projects listed here are defined along these projects. Luckily we did not have to start from total scratch: Daniel Hautzinger, one of our more senior team members, was doing his diploma thesis in the lab of Kristian Müller working with DNA origami while we were also in the lab and Wolfgang Schamel could provide the mentioned T-cell line with a TCR fused to a scFv and a B-cell line directed towards the same antigen.

Subprojects

Modeling
3D-Modeling
DNA-Origami
Cloning Strategy
Cell Culture
Transfection and Synthetic Receptor Activation
Cell Stability, Ca2+ Signaling and DNA-Origami-Binding

Highlights

Modeling
The dimerization of the extracellular receptor domains is a important necessity for the functionality of our Modular Synthetic Receptor System. Presenting the system a stimulus in the form of spatial arranged ligands (nitro-iodo-phenol or fluorescein molecules), the extracellular domains dimerize, thus the corresponding intracellular parts such as the split lactamase halves or split fluorescent proteins complement to measureable output. To analyse the theoretical functionality due to dimerization, two receptor dimerization models (one T cell receptor model and one general receptor model) are introduced and discussed and a proper model for the Modular Synthetic Receptor System is constructed. It consists of 10 reaction kinetic equations, 9 ordinary differential equations including 25 variable parameters. Matlab m-files are embedded for further inspection.

Split protein activity in time dependent on ligand amount

structural model of expression part Bba_K157037

3D-Modeling
We have created various three-dimensional models of our constructs using Pymol and SwissPdbViewer.
Pdb-files of the huge DNA-Origami molecule were created as adequate as possible using Nano-Engineer version 1.0 and then edited in Pymol. These Models were used to plan the spatial arrangement and, thus, input-pattern of antigens at a nanometer scale.

DNA-Origami
The creation of DNA-Origami was one of the first subprojects we engaged in. Structural planing of the input pattern on the Origami-surface and order of the modified oligonucleotides were the first issues, increasing of the Origami-yield and measurement of the molecule´s stability in cell culture media the latter ones.

AFM-measurement of DNA-Origami

cloning scheme

Cloning Strategy
All of our constructs were cloned successfully using our extended pre- and suffix and plasmid "pMA" (part Bba_K157000). The broad combinatorial range given by the modular concept was almost fully exploited, so that we ended up with the submission of 13 basic and 28 composite parts.
Once a construct was completed, it was cloned into the CMV-promoted transfection vector (part Bba_K157040) and used for transfection of 293T-cells.

Transfection and Synthetic Receptor Activation
Transfection of 293T-cells has also been shown to work for most of our fusion proteins; so far, we could even show that at least our constructs with lipocalin FluA as extracellular domaine are integrated into the membrane.
All transfections were carried out with part Bba_K157040, one of our composite parts consisting of the transfection vector Bba_J52017 and a CMV-promotor. Due to the limited time range we were not able to activate/dimerize any of the various possible "receptor"-pairs yet, but we are hoping to receive a positive result in that concern until the jamboree.

membrane-localization of Part Bba_K157032-YFP

Ca-influx measurement

Cell Stability, Ca2+ Signaling and DNA-Origami-Binding
Due to the small total amounts of DNA-Origami we could not use FACS to measure calcium influx and, thus, T-cell activation. The alternative we found were "Nano-Slides" for inverse fluorescence microscopy; T-cells were immobilized on the poly-L-Lysine coated slides and stimulated directly under the microscope. This procedure allowed real-time observation of calcium-influx.

Literature

Split-fluorophores:
-Chang-Deng Hu, Yurii Chinenov, Tom K. Kerppola: ”Visualization of Interactions among bZIP and Rel Family Proteins in Living Cells Using Bimolecular Fluorescence Complementation”, Molecular Cell, Vol. 9, 789–798, April, 2002
-Chang Deng Hu, Tom K. Kerppola: “Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis”, Nat Biotechnol. 2003 May; 21(5):539-545 (doi:10. 1038/nbt816)
-Tom K. Kerppola: “Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells”, Nat Protoc. 2006;1(3):1278-1286 (doi:10.1038/nprot.2006.201)
-Nagai, T. et al. “A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications” J. Biol. Chem. 276, 29188-29194, 2001
-Roger Y. Tsien et al. „Creating new fluorescent probes for cell biology“, Nature Biotechnology Reviews, Vol. 3, 906-918, 2002
LipocalinFluA:
-Gerald Beste, Frank S. Schmidt, Thomas Stibora and A. Skerra: “Small antibody-like proteins with prescribed ligand specificities derived from the lipocalin fold“,Proc. Natl. Acad. Sci. USA Vol. 96, pp. 1898–1903, March 1999 Biochemistry
-Ingo P. Korndörfer, Gerald Beste and A. Skerra: “Crystallographic Analysis of an “Anticalin” With Tailored Specificity for Fluorescein Reveals High Structural Plasticity of the Lipocalin Loop Region”, PROTEINS: Structure, Function, and Bioinformatics 53:121–129 (2003)
DNA-Origami:
Paul W. K. Rothemund: Nature 440, 297-302 (16 March 2006)
Antibody B1-8:
-Ana Cumano and Klaus Rajewski: “Clonal recruitment and somatic mutation in the generation of immunological memory to the hapten NP”, The EMBO Journal vol. 5 no.10 pp. 2459-2468, 1986
-D. Allen, T. Simon, F. Sablitzky, K. Rajewski and A. Cumano: “Antibody engineering for the analysis of affinity maturation of an anti-hapten response”, The EMBO Journal vol. 7 no.7 pp. 1995-2001, 1988
Modeling:
-Martin F. Bachmann, Michael Salzmann, Annette Oxenius and Pamela S. Ohashi: "Formation of TCR dimers/trimers as a crucial step for T cell activation", Eur. J. Immunol., 1998
-Martin F. Bachmann and Pamela S. Ohashi: "The role of T-cell receptor dimerization in T-cell activation", Review Immunology Today, Dezember 1999
-João Sousa and Jorge Carneiro: "A mathematical analysis of TCR serial triggering and down-regulation", Eur. J. Immunol., 2000
TCR activation:
-Susana Minguet, Mahima Swamy, Balbino Alarcón, Immanuel F. Luescher and Wolfgang W.A. Schamel: "Full Activation of the T Cell Receptor requires Both Clustering and Conformational Changes at CD3", Immunity, 2006

@@ Line 5: / Line 5: @@
 <br>
 <br>
-<h2>'''Introduction'''</h2>
+<h2>'''Summary'''</h2>
-This year´s main project is the attempt to create an "artificial receptor-system", featuring extra- and intracellular modules as well as suitable transmembrane regions.
+'''Modular Synthetic Receptor System'''<br>
-The intracellular domaine of our receptor-device is build by halves of split reporter-proteins that can reassemble and will then produce readable output, e. g. fluorescence.
-Each one of these protein-halves is connected to its extracellular domaine by a single-span transmembrane-helix.
+In the following we provide a summary of our project and display the highlights of our achievements. For a more detailed view please see the reports of each sub-project.<br>
-The extracellular or detecting domaine consists of a protein or peptide with the ability to bind a certain molecule.<br>
+The general goal of the Freiburg 2008 team is to establish a synthetic transmembrane receptor system comprising the extracellular input devices, the extracellular receiver domain, the transducer to the cytosol and the reporter/executor in the cytosol.<br>
-Now, if a system with two matching receptors is presented these molecules in a strict, pairwise spatial arrangement, the receptor-devices are brought together,
+As controllable receptor activation event, we chose spatial control of dimerization, which is a mechanism often used by nature. To achieve spatial control in the nanometer range of proteins we chose DNA origami for exact and complex patterns with several ligands and chemical coupling of one type of ligand to scaffold proteins for simple multimerization.<br>
-the split reporter-protein reassembles inside the cell and the output can be detected.
+To facilitate binding, we used a single-chain Fv fragment and a designed anticalin. Both bind haptens which can be conjugated to the DNA-Origami or scaffold proteins. Transduction to the cytosole is mediated by a single transmembrane helix.<br>
-We employ so-called "Origami-DNA" to create the exactly defined molecule-patterns that are needed to activate our receptors.
+As intracellular reporters we employ split proteins. Here, we used the split lactamase from the iGEM2007 Freiburg project as well as the split fluorescent proteins CFP/cerulean and YFP/venus. To provide steric flexibility and reach of our binding and reporting modules we interspersed linkers between each functional domain.<br>
-<br>
+All parts are cloned in in E. coli and feature full BioBrick-compatibility. However, we test our receptors in human or mouse cells/chassis. Thus, to direct our constructs to the cell surface we attached a eukaryotic signal peptide to the N-terminus of each synthetic receptor. In addition to the new synthetic receptors, we also employed an existing T-cell line displaying a TCR fused to a single chain as a test for the spatial control.<br>
-One of the main inspirations that lead to the idea of creating a synthetic receptor-like fusion protein is based on an immunologic study on the signaling pathway of the T-Cell-Receptor (TCR) that has been performed by Wolfgang Schamel at the Max-Planck-Institute for immunology, Freiburg.
+Besides designing and cloning parts we spent quite some time on establishing and analyzing the biological experiment. In particular we wanted to test the spatial control via DNA origami using an existing cell line expressing the scFv fused to a T-cell receptor. Here we addressed the following questions:<br><br>
-In this study he used modified TCRs with Fab-Fragment-singlechains of Anti-NIP –Antibodies fused to their ß-domaines by a flexible linker that would present them on the cell´s surface.
+- Can we improve DNA origami assembly yield by varying the staple oligonucleotide to scaffold DNA ratio?<br>
-This modification would allow to investigate the influence of receptor-clustering on the intensity of the cell-signaling. It could been shown that there is a relation between the clustering of the antigen and, thus, of the receptors by presenting various peptides with certain amounts and arrangements of NIP-molecules as stimulus.<br>
+- Can we find buffer conditions, which mediate cell viability and origami stability?<br>
-Anyway, this experiment was restricted by the one-dimensionality of the antigen-fused peptides; at this point, the Origami-DNA comes into play:
+-Can we downsize a Ca2+ release assay to fit the sample sizes of our precious DNA origami?<br>
-Paul Rothemund had discovered that it is possible to shape M13-Phage single-strand-DNA simply adding oligonucleotides that would work as „brackets“ when complementing the long single-strand. In this way, one can generate DNA-squares of a certain size with „nods“ at certain distances.<br>
+- Do our constructs express and then localize in the membrane of eukaryotic cells?<br>
-One member of our team, Daniel Hautzinger, has recently finished his diploma-thesis on Origami-DNA and the possibilities of generating patterns on these square surfaces by modifying the  Oligo-nucleotides that build up the nod-points.
+-Can we detect synthetic receptor activation?<br><br>
-As the antigen NIP can as well be fused to these oligos, it was now possible to present strictly defined two-dimensional antigen-patterns to T-Cells carrying the modified receptors mentioned above.<br>
+To reach our goal within the short given time frame we started several subprojects in parallel. Our sub-projects listed here are defined along these projects. Luckily we did not have to start from total scratch: Daniel Hautzinger, one of our more senior team members, was doing his diploma thesis in  the lab of Kristian Müller working with DNA origami while we were also in the lab and Wolfgang Schamel could provide the mentioned T-cell line with a TCR fused to a scFv and a B-cell line directed towards the same antigen.
-This, again, made us come up with the idea of a transmembrane-fusion-protein that could be spatially arranged from outside the cell by the pattern on the Origami-DNA-surface.<br>
-Of course, the first extracellular domaine we had in mind was the anti-NIP-singlechain Schamel had used with his receptors. The first intracellular domaines should consist of the split-lactamase-halfes we designed as parts for last year´s iGEM, as this enzyme´s activity can be regained by complementation of the halves and detected by a fluorescent substrate.
-Now, we were looking for a single-span-transmembrane-protein; as the domaines of the Epidermal-Growth-Factor Receptor are well known, we chose to employ it´s transmembrane-helix and the signal-peptide mediating the construct´s insertion into the membrane.<br>
-Further modules we had in mind were an Anti-Fluorescein-singlechain and a fluorescein-binding variety of Lipocalin by Arne Skerra as extracellular „detectors“ as well as the complementing halves of each one of the split-fluorophores „Cerulean“ (cyan) and „Venus“ (yellow) as intracellular „reporters“. These split-fluorophores feature cross-compatibility  between the N- and C-terminal halves (green fluorescence), enabling our system to generate three different „outputs“ (yellow, blue, green) with only two molecules (NIP, FluA) building up the „input-pattern“ on the Origami-DNA-surface.
 <br><br>
 <h2>'''Subprojects'''</h2>