# Team:ETH Zurich/modeling/diffmodel

(Difference between revisions)
 Revision as of 18:19, 17 October 2014 (view source)Eledieu (Talk | contribs)m (→Initial conditions determined by bead preparation step)← Older edit Latest revision as of 01:13, 18 October 2014 (view source)Clormeau (Talk | contribs) (→Pattern developing) (9 intermediate revisions not shown) Line 15: Line 15: === Geometry and boundary conditions === === Geometry and boundary conditions === - [[File:ETH Zurich Compartments Diffusion.png|float|500px|thumb|Millifluidic compartments used for the diffusion model]] + [[File:ETH Zurich Compartments Diffusion.png|float|500px|thumb|'''Figure 1''' Millifluidic compartments used for the diffusion model]] Line 30: Line 30: - [[File:ETHZ comsol geometry.png|center|610px|thumb|Geometry used for simulation on Comsol Multiphysics]] + [[File:ETHZ comsol geometry.png|center|610px|thumb|'''Figure 2''' Geometry used for simulation on Comsol Multiphysics]]

Line 167: Line 167: The cells of the first row are induced by 10 μ M AHL just before they are encapsulated in alginate. Then, bead formation takes half-an-hour before these cells are added in the first well. During this half-an-hour AHL can diffuse out. Therefore initial conditions in the first cell completely depend on these previous steps. We have also simulated this bead preparation in order to get these initial conditions. The cells of the first row are induced by 10 μ M AHL just before they are encapsulated in alginate. Then, bead formation takes half-an-hour before these cells are added in the first well. During this half-an-hour AHL can diffuse out. Therefore initial conditions in the first cell completely depend on these previous steps. We have also simulated this bead preparation in order to get these initial conditions. - [[File:ETHZ Beads in storage solution.jpg|right|300px|thumb|Beads stored in calcium chloride, 10mM]] + [[File:ETHZ Beads in storage solution.jpg|center|300px|thumb|'''Figure 3''' Beads stored in calcium chloride, 10mM]] The geometry is a simple bead in a bigger compartment filled with calcium chloride. We look at the concentrations at the middle of the bead. Initial concentration of LuxR before bead diffusion starts is a classical steady state aLuxR/dLuxR The geometry is a simple bead in a bigger compartment filled with calcium chloride. We look at the concentrations at the middle of the bead. Initial concentration of LuxR before bead diffusion starts is a classical steady state aLuxR/dLuxR - [[File:ETHZ Signaling molecules.png|600px|center]] + [[File:ETHZ Signaling molecules.png|600px|center|thumb|'''Figure 4''' Signaling molecules]] - [[File:ETHZ Regulators and GFP.png|600px|center]] + [[File:ETHZ Regulators and GFP.png|600px|center|thumb|'''Figure 5''' Regulators and GFP]] Extracellular AHL diffuses very fast through the membrane, which makes intracellular AHL increase, until both reach the same value and diffusion through the membrane reaches an equilibrium. From this point on AHL starts to bind to LuxR to form RLux, so AHL decreases, until RLux triggers enough production of LuxI which catalyses again AHL production, and makes AHL concentration become stable. Extracellular AHL diffuses very fast through the membrane, which makes intracellular AHL increase, until both reach the same value and diffusion through the membrane reaches an equilibrium. From this point on AHL starts to bind to LuxR to form RLux, so AHL decreases, until RLux triggers enough production of LuxI which catalyses again AHL production, and makes AHL concentration become stable. Line 219: Line 219: The background in our experimental setup is very high and this is high we see actual fluorescence appear only after 11 hours. In order to account for this, we also set up a background in the simulated pattern by adjusting the scale. The background in our experimental setup is very high and this is high we see actual fluorescence appear only after 11 hours. In order to account for this, we also set up a background in the simulated pattern by adjusting the scale. - ------------ Video + - -------------------- + {|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto; font-size:10pt;" + |colspan="2" style='font-size:10pt';text-align:left|{{:Team:ETH_Zurich/Templates/Video|width=1080px|id=video3|ratio=1920/720|srcMP4=http://2014.igem.org/wiki/images/b/b1/ETH_Zurich_2014_signal_propagation_with_simulation.mp4|poster=http://2014.igem.org/wiki/images/6/69/ETH_Zurich_2014_signal_propagation_with_simulation_preview.png}} + |- + |colspan="2" style='font-size:10pt';text-align:left|'''Video 1''' '''Row wise, self-propagating [http://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing cell-to-cell communication] of ''E. coli'' cells confined in [http://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] (d=3 mm, initially 107 cells/bead) on a [http://2014.igem.org/Team:ETH_Zurich/lab/chip custom-made millifluidic PDMS chip].''' + |- + |style="width:50%"|All fluorescent cells contained [http://2014.igem.org/Team:ETH_Zurich/expresults/rr#Riboregulators riboregulated] sfGFP followed by [http://parts.igem.org/Part:BBa_C0161 LuxI (BBa_C0161)] together under the control of the [http://parts.igem.org/Part:BBa_R0062 pLux promoter (BBa_R0062)], and [http://parts.igem.org/Part:BBa_J23100 constitutively (BBa_J23100)] expressed [http://parts.igem.org/Part:BBa_C0062 LuxR (BBa_C0062)]. LuxI catalyzes the production of the autoinducer 3OC6-HSL, which is then diffusing from cell to cell. For initialization, the cells in one bead of the top row were induced with 3OC6-HSL before encapsulation. Imaging was implemented with a [http://2014.igem.org/Team:ETH_Zurich/lab/protocols#Biostep_Dark-Hood_DH-50.E2.84.A2__and_the_Argus-X1.E2.84.A2_software Biostep Dark-Hood DH-50 (Argus X1 software)] fitted with a Canon EOS 500D DSLR camera and a fluorescence filter (545 nm filter). Pictures were usually taken every 2 min at an excitation wavelength of 470 nm with the standard Canon EOS Utility software. Time-lapse movies were created with Adobe After Effects CC software. 1950x faster than real-time, the video shown starts 10 h after the initiation of the experiment (however the time scale shown corresponds to minutes after loading of the chip). For precise experimental setup, check the [http://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion Results] page. + ||Simulation of the propagation of the pattern in the millifluidic chip. [http://www.comsol.com/comsol-multiphysics Comsol Multiphysics Simulation software] was used in order to simulate a detailed diffusion model including quorum sensing steps in colonies and cell growth. Overall GFP concentration in beads has been scaled in order to account for the high background of the experimental setup. Green Fluorescence Protein is produced earlier in the wells, but can be seen only above a certain threshold.
Accurate prediction of experimental data by the model has been achieved, with parameters from our own fittings or from the literature. Experimental observation combined with simulation enables to show that a pattern is able to develop in the millifluidic chip in a reasonable time scale. + |} === Dynamics by row === === Dynamics by row === Line 228: Line 235:

- [[File:ETHZ AHL-regulators-pattern.png|center]] + [[File:ETHZ AHL-regulators-pattern.png|center|900px|thumb|'''Figure 6''']]

Line 236: Line 243:

- [[File:ETHZ GFP in beads.png|600px|center]] + [[File:ETHZ GFP in beads.png|600px|center|thumb|'''Figure 7''' GFP in beads ]] - [[File:ETHZ GFP in single cell.png|600px|center]] + [[File:ETHZ GFP in single cell.png|600px|center|thumb|'''Figure 8''' GFP in single cell]]

## Latest revision as of 01:13, 18 October 2014

iGEM ETH Zurich 2014