Team:ETH Zurich/lab/chip

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==Overview==
==Overview==
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Our project aims for the biological implementation of cellular automata, so we had to find a way to create a regular grid of cells with a defined neighborhood as shown in the pictures below. On the left side a classical cellular automata is depicted, on the right side an outline of our biological version consisting of a grid-like polydimethylsiloxane (PDMS) chip filled with cell colonies encapsulated in alginate beads.
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Our project aims for the biological implementation of [https://2014.igem.org/Team:ETH_Zurich/project/background/modeling#Cellular_Automata cellular automata], so we had to find a way to create a regular grid of cells with a defined neighborhood as shown in the figures below. On the left side a classical cellular automata is depicted (see figure 1), on the right side an outline of [https://2014.igem.org/Team:ETH_Zurich/project/overview#Implementation_in_E._coli our biological version] consisting of a grid-like polydimethylsiloxane (PDMS) chip filled with [https://2014.igem.org/Team:ETH_Zurich/lab/bead cell colonies encapsulated in alginate beads] (see figure 2).
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[[File:ETH Zurich Rule 6.PNG|200px|thumb|left|Classical grid from cellular automata theory (ON state=back, OFF state=white).]]
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[[File:ETH Zurich Rule 6.PNG|300px|thumb|left|'''Figure 1''' '''Classical grid from cellular automata theory''' (ON state=back, OFF state=white).]]
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[[File:ETH_Zurich_2014_theoretical_grid.png|210px|thumb|left|Outline of a PDMS-made grid loaded with cells confined in alginate beads for the biological implementation of cellular automata (ON state=sfGFP/green, OFF state=white).]]
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[[File:ETH_Zurich_2014_theoretical_grid.png|300px|thumb|right|'''Figure 2 Outline of a PDMS-made grid loaded with cells confined in alginate beads for the biological implementation of cellular automata''' (ON state=sfGFP/green, OFF state=white).]]
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In the following, we have investigated the combination of additive manufacturing (3D-printing) and PDMS chip fabrication for applications in synthetic biology. This rapid prototyping approach allowed us to update our chips continuously according to new insights from modeling or the wet lab and in particular to avoid more intricate photolitographic approaches, which generally require clean room access, relatively expensive raw materials, and in depth knowledge of etching techniques.
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In the following, we have investigated the combination of additive manufacturing (3D-printing) and PDMS chip fabrication for applications in synthetic biology. This rapid prototyping approach allowed us to update our chips continuously according to new [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel insights from modeling] or the wet lab and in particular to avoid more intricate photolitographic approaches, which generally require clean room access, relatively expensive raw materials, and in depth knowledge of etching techniques.
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As a result, we are convinced that the tinkering with 3D-printing for mold creation is more economical for our applications and perfectly in line with the do-it-yourself spirit of iGEM.
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As a result, we are convinced that the tinkering with 3D-printing for mold creation is more economical for our applications and measurements. Also it is perfectly in line with the do-it-yourself spirit of iGEM.
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===Mold Design and 3D Print Exchange===
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==Mold Design and 3D Print Exchange==
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Our custom-made plates and molds were design using a common personal computer (MacBook Air, 13-inch, early 2014, 1.7 GHz Intel Core i7, 8 GB 1600 MHz) and a 3D computer aided design (CAD) software package that is freely available for Mac OS X 10.9.4 ([http://www.123dapp.com/design Autodesk123D Design]). The CAD models were exported as mesh files (.stl) to the 3D printer's software ([http://www.makerbot.com/support/makerware/troubleshooting/ MakerWare]). The dimensions of the device-structures were usually between 1 mm and 5 mm, falling in the range of millifluidics.   
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Our custom-made plates and molds were design using a common personal computer (MacBook Air, 13-inch, early 2014, 1.7 GHz Intel Core i7, 8 GB 1600 MHz) and a 3D computer aided design (CAD) software package that is freely available for Mac OS X 10.9.4 ([http://www.123dapp.com/design Autodesk123D Design]). The CAD models were exported as mesh files (.stl) to the 3D printer's software ([http://www.makerbot.com/support/makerware/troubleshooting/ MakerWare]). The dimensions of the device-structures were usually between 1 mm and 5 mm, falling in the range of millifluidics<sup>[[Team:ETH_Zurich/project/references#refKitson|[31]]]</sup>.   
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{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"
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File:ETH_Zurich_mesh_2_20140826.jpg|b.
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|[[File:ETH_Zurich_mesh_2_20140826.jpg|200px]]
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File:ETH_Zurich_mesh_3_20140826.jpg|c.
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|[[File:ETH Zurich 2014 final mold model 2.jpeg|200px]]
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File:ETH Zurich 2014 2 chamber connected plate model.jpeg|g.
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|[[File:ETH Zurich 2014 diffusion plate model.jpeg|200px]]
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File:ETH Zurich 2014 diffusion plate model.jpeg|h.
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|'''Figure 3-a''' The first design for a gel-comb, a mold for a millifluidic PDMS chip and a corresponding box for the mold.
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|'''Figure 3-b''' The final mold design for our millifluid PDMS chip used for [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing cell-to-cell communication] experiments.
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|'''Figure 3-c''' A design for a 96-well plate with connected wells, which allows automated measurements in a plate reader.
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<gallery mode="traditional" widths=200px>
 
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File:ETH_Zurich_mesh_2_20140826.jpg|b.
 
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File:ETH_Zurich_mesh_2_20140826.jpg|b.
 
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File:ETH Zurich 2014 final mold model 2.jpeg|f.
 
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File:ETH Zurich 2014 final mold model.jpeg|e.
 
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'''All mesh files designed during the project will be made available at the [http://3dprint.nih.gov/ NIH 3D Print Exchange] under the category 'Custom Labware' via our [http://3dprint.nih.gov/users/ethzurichigem2014 ETH_Zurich_iGEM2014] account.
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'''
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===3D-Printing and Rapid Prototyping===
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==3D-Printing and Rapid Prototyping==
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The mold designs were printed with a commercial 3D-printer (2nd generation MakerBot with MakerWare software; MakerBotIndustries, Brooklyn, US; 5th generation US$2'899) with acrylonitrile butadiene styrene (ABS, a copolymer of acrylonitrile, butadiene, and  
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The mold designs were printed with a commercial 3D-printer (2nd generation MakerBot Replicator with MakerWare software; [http://www.makerbot.com MakerBotIndustries], Brooklyn, US; 5th generation US$2'899) with acrylonitrile butadiene styrene ([http://en.wikipedia.org/wiki/Acrylonitrile_butadiene_styrene ABS], a copolymer of acrylonitrile, butadiene, and styrene). The maximum object size printable is [mm]: 225 x 145 x 150. The precision and minimum feature size are given as [mm]: 0.011 (XY-axis), 0.0025 (Z-axis); and 0.4 (XY-axis), 0.2 (Z-axis) respectively. The printing time varied with the size of the mold but was usually below 4 hours.  
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styrene). The maximum object size printable is [mm]: 225 x 145 x 150. The precision and minimum feature size are given as [mm]: 0.011 (XY-axis), 0.0025 (Z-axis); and 0.4 (XY-axis), 0.2 (Z-axis) respectively. The printing time varied with the size of the mold but was usually below 4 hours.  
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File:ETH Zurich 2014 MakerBot.jpg|j.
 
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File:ETH Zurich 2014 MakerWare.jpg|i.
 
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File:ETH Zurich 2014 MakerBot ABS.jpg|k.
 
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All fabricated structures were ready to use after removing the support structures and did not require additional surface treatments like sonication, curing, painting or silanization. The molds were then directly used for PDMS chip production. In addition, custom made black 96-well plates (connected wells for diffusion assays, plate reader compatible) were printed but found to be leaky over time. The material costs of the molds were in the range of US$2 to US$4 and for the 96-well plates below US$8 (about US$160 per kg of ABS). The maximum resistance to continuous heat is given as 90 degrees Celsius<sup>[[Team:ETH_Zurich/project/references#refCRC|[23]]]</sup>, as a result autoclaving at 121 degrees Celsius was not feasible (see picture below).
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|[[File:ETH Zurich 2014 MakerBot.jpg|200px]]
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|[[File:ETH Zurich 2014 MakerWare.jpg|200px]]
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|[[File:ETH Zurich 2014 MakerBot ABS.jpg|x200px]]
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|'''Figure 4-a''' The MakerBot Replicator (2nd generation) we used to print our molds.
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|'''Figure 4-b''' 'Screenshot' of the MakerWare software we used to print our molds.
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|'''Figure 4-c''' A roll of ABS filament used by the 3D-printer.
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File:ETH Zurich 2014 box autoclaved.JPG|o.
 
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File:ETH Zurich 2014 small grid.JPG|q.
 
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File:ETH Zurich diffusion plate.JPG|r.
 
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File:ETH Zurich 2014 96 well all connected.jpeg|l.
 
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All fabricated structures were ready to use after removing the support structures and did not require additional surface treatments like sonication, curing, painting or silanization. The molds were then directly used for PDMS chip production. In addition, custom made black 96-well plates (connected wells for diffusion assays, plate reader compatible) were printed but found to be leaky over time. The material costs of the molds were in the range of US$2 to US$4 and for the 96-well plates below US$8 (about US$160 per kg of ABS). The maximum resistance to continuous heat is given as 90 ⁰C <sup>[[Team:ETH_Zurich/project/references#refCRC|[23]]]</sup>, as a result autoclaving at 121 ⁰C was not feasible and led to deformation (see the box in figure 5-a).
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File:ETH Zurich 2014 diffusion mold.JPG|p.
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File:ETH Zurich 2014 diffusion mold.JPG|p.
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File:ETH Zurich 2014 final mold.jpeg|m.
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File:ETH Zurich 2014 final mold closeup.jpeg|n.
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{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"
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|[[File:ETH_Zurich_2014_comb_and_box.jpg|200px]]
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|[[File:ETH Zurich 2014 small grid.JPG|200px]]
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|[[File:ETH Zurich diffusion plate.JPG|200px]]
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|[[File:ETH Zurich 2014 96 well all connected.jpeg|200px]]
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|'''Figure 5-a''' Printed gel-comb and box. The box was autoclaved at 121 ⁰C.
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|'''Figure 5-b''' Printed millifluid grid with interconnected wells (edge length of 3 mm).
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|'''Figure 5-c''' Printed 96-well plate, pairs of wells (edge length of 5 mm) are connected by channels of varied length (1 mm to 6 mm).
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|'''Figure 5-d''' Printed 96-well plate, all wells (edge length of 5 mm) are connected.
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===PDMS Chip Preparation===
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==PDMS Chip Preparation==
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For the fabrication of millifluidic-chips raw PDMS (Dow Corning Sylgard 184) was prepared by mixing base and curing agent in 10:1 proportion. The PDMS solution was mixed vigorously and degassed (desiccator connected to vacuum) until no  further bubble formation could be observed. Subsequently the mixture was poured over the mold and cured in an vacuum oven over night. The PDMS chip was easily removed without additional aids and placed in clear plastic trays (86 x 128 mm; OmniTrays, Thermo Scientific). The wells were then filled with CB medium and loaded with cells encapsulated in alginate beads.
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For the fabrication of millifluidic-chips raw PDMS (Dow Corning Sylgard 184) was prepared by mixing base and curing agent in 10:1 proportion. The PDMS solution was mixed vigorously and degassed (desiccator connected to vacuum) until no  further bubble formation could be observed. Subsequently the mixture was poured over the mold and cured in an vacuum oven over night at RT. While the first mold design separated insufficiently from the PDMS due to an inappropriate aspect ratio (see figures 6-a and 7-a), all other PDMS chips were easily removed without additional aids and placed in clear plastic trays (86 x 128 mm; OmniTrays, Thermo Scientific).  All the mold shown below were at least used once before the pictures were taken. The PDMS wells were then filled with [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#Complex_bead_medium_.28CB_medium.29 CB medium] and loaded with cells encapsulated in [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads].
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File:ETH Zurich 2014 broken chip.jpeg|s.
 
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File:ETH Zurich 2014 2 well diffusion chip.jpeg|t.
 
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File:ETH Zurich 2014 2 well diffusion chip upsidedown.jpeg|u.
 
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File:ETH Zurich 2014 PDMS diffusion chip final.jpeg|v.
 
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{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"
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|[[File:ETH_Zurich_2014_first_mold_with_PDMS.jpg|200px]]
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|[[File:ETH Zurich 2014 diffusion mold.JPG|200px]]
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|[[File:ETH Zurich 2014 final mold.jpeg|200px]]
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|[[File:ETH Zurich 2014 final mold closeup.jpeg|200px]]
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|'''Figure 6-a''' The very first mold design. PDMS stuck between the wells while removing it.
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|'''Figure 6-b''' Mold design for a diffusion assay with two connected chambers (edge length of 4 mm) with varied channel length (1 mm to 4 mm).
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|'''Figure 6-c''' The final mold design for [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing cell-to-cell communication] experiments (edge length of 5 mm, channel length of 3 mm).
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|'''Figure 6-d''' Close up of the final mold design. The separate layers are clearly visible ('additive' manufacturing).
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{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"
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|[[File:ETH Zurich 2014 broken chip.jpeg|200px]]
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|[[File:ETH Zurich 2014 2 well diffusion chip upsidedown.jpeg|200px]]
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|[[File:ETH Zurich 2014 PDMS diffusion chip final.jpeg|200px]]
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|[[File:ETH_Zurich_2014_final_chip_zoom.png|200px]]
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|'''Figure 7-a''' The very first PDMS chip. As the close-up shows, the outer parts are well defined, but the middle part did not separate from the mold due to an inappropriate aspect ratio.
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|'''Figure 7-b''' PDMS chip for diffusion assays with two connected chambers (edge length of 4 mm) and varied channel length (1 mm to 4 mm).
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|'''Figure 7-c''' The final PDMS chip for [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing cell-to-cell communication] experiments (edge length of 5 mm, channel length of 3 mm).
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|'''Figure 7-d''' Close up of the final PDMS chip. The channels are well defined and even small structures separated evenly from the mold.
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==Time-Lapse Movies==
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Below you find an overview of the time-lapse movies taken during the summer. In the very first trial the wells were filled with [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#LB_medium_from_dehydrated_product LB agar], holes were punched with a pipette tip and filled with highlighter-ink ([http://en.wikipedia.org/wiki/Pyranine pyranine]) to visualize diffusion (see video 1). Later, different set-ups were tested: chambers filled with liquid [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#LB_medium_from_dehydrated_product LB medium] separated by solidified 2% agarose in the connecting channel and [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] in liquid [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#Complex_bead_medium_.28CB_medium.29 CB medium]. We continued with the 'alginate beads in  liquid medium' set-up, as it yielded the most promising intermediate results, and could then finally show cell-to-cell communication of bacteria confined in beads on our millifluid chip.
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In all videos shown imaging was implemented with a [https://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 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.
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===Time-Lapse Movies===
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|{{:Team:ETH_Zurich/Templates/Video|width=300px|id=video1|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/c/c7/ETH_Zurich_2014_two_wells_1st_test_with_highlighter.mp4</html>}}
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|{{:Team:ETH_Zurich/Templates/Video|width=300px|id=video2|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/1/18/ETH_Zurich_2014_two_wells_liquid_culture_small.mp4</html>}}
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|'''Video 1 The very first diffusion experiment with fluorescent highlighter ink ([http://en.wikipedia.org/wiki/Pyranine pyranine]).''' The wells of the PDMS chip were filled with [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#LB_medium_from_dehydrated_product LB agar]. About 5 μL ink were added in a punched whole on one side of the two wells. ~4500x faster than real-time.
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|'''Video 2 Diffusion experiment with liquid cultures.''' The wells of the PDMS chip were filled with [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#LB_medium_from_dehydrated_product LB medium], separated by solidified 2% agarose in the channel. The bottom well contained 3OC6-HSL (~1 mM), the top well ''E. coli'' cells with sfGFP under the control of pLux. ~4500x faster than real-time.
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|{{:Team:ETH_Zurich/Templates/Video|width=300px|id=video3|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/7/7d/ETH_Zurich_2014_AHL_bead_sensor_bead.mp4</html>}}
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|{{:Team:ETH_Zurich/Templates/Video|width=300px|id=video4|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/8/8c/ETH_Zurich_2014_sender_receiver_beads_small.mp4</html>}}
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|'''Video 3  Diffusion experiment with [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] in defined liquid medium.''' The wells of the PDMS chip were filled with CB medium. The bottom well contained beads with 3OC6-HSL (~1 mM), the top well ''E. coli'' cells with [https://2014.igem.org/Team:ETH_Zurich/lab/sequences#Sensor_Constructs sfGFP under the control of pLux] confined in [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] (d=3 mm, intially 10<sup>7</sup> cell/beads). ~1850x faster than real-time.
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|'''Video 4 Cell communication experiment with [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] in defined liquid medium.''' The wells of the PDMS chip were filled with CB medium. The bottom well contained ''E. coli'' [https://2014.igem.org/Team:ETH_Zurich/lab/sequences#Producer_Constructs cells expressing LuxI], which catalyzes the production of 3OC6-HSL; the top well contained ''E. coli'' cells with [https://2014.igem.org/Team:ETH_Zurich/lab/sequences#Sensor_Constructs sfGFP under the control of pLux]. All cells were confined in [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] (d=3 mm, intially 10<sup>7</sup> cell/beads). ~3450x faster than real-time.
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|colspan="2"|{{:Team:ETH_Zurich/Templates/Video|width=600px|id=video5|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/a/a9/ETH_Zurich_2014_signal_propagation.mp4</html>}}
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|colspan="2"|'''Video 5 Row wise, self-propagating [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing cell-to-cell communication] of ''E. coli'' cells confined in [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] (d=3 mm, intially 10<sup>7</sup> cell/beads) on a [https://2014.igem.org/Team:ETH_Zurich/lab/chip custom-made millifluidic PDMS chip].''' All cells contained [https://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. 1750x faster than real-time, the video starts 7 h after the initiation of the experiment.
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Imaging was implemented with a 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 (470 nm) with the standard Canon EOS Utility software. Time-lapse movies were created with Adobe After Effects CC software.
 
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Below you find the time-lapse movies taken during the summer. In the very first trial the wells were filled with LB agar, holes were punched with a pipette tip and filled with highlighter-ink (pyranine) to visualize diffusion. Later, different set-ups were tested: chambers filled with liquid medium separated by solidified 2% agarose in the connecting channel and alginate beads in liquid medium.
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We continued with the 'alginate beads in  liquid medium' set-up, as it yielded the most promising intermediate results, and could then finally show cell-to-cell communication of bacteria confined in beads on our millifluid chip.
 

Latest revision as of 03:27, 18 October 2014

iGEM ETH Zurich 2014