http://2014.igem.org/wiki/index.php?title=Special:Contributions/Mbahls&feed=atom&limit=50&target=Mbahls&year=&month=2014.igem.org - User contributions [en]2024-03-28T14:04:44ZFrom 2014.igem.orgMediaWiki 1.16.5http://2014.igem.org/Team:ETH_Zurich/lab/chipTeam:ETH Zurich/lab/chip2014-10-18T03:23:49Z<p>Mbahls: /* Time-Lapse Movies */</p>
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<div>{{:Team:ETH_Zurich/tpl/head|Millifluidic Chip & Rapid Prototyping}}<br />
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<html><article style='min-height:800px'></html><br />
==Overview==<br />
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).<br />
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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).]]<br />
[[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).]]<br />
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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.<br />
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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.<br />
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<html><article id='Mold'></html><br />
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==Mold Design and 3D Print Exchange==<br />
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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>. <br />
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{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_mesh_2_20140826.jpg|200px]]<br />
|[[File:ETH Zurich 2014 final mold model 2.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion plate model.jpeg|200px]]<br />
|-<br />
|'''Figure 3-a''' The first design for a gel-comb, a mold for a millifluidic PDMS chip and a corresponding box for the mold.<br />
|'''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. <br />
|'''Figure 3-c''' A design for a 96-well plate with connected wells, which allows automated measurements in a plate reader.<br />
|}<br />
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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.<br />
'''<br />
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<html><article id='3D'></html><br />
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==3D-Printing and Rapid Prototyping==<br />
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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. <br />
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{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 MakerBot.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerWare.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerBot ABS.jpg|x200px]]<br />
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|'''Figure 4-a''' The MakerBot Replicator (2nd generation) we used to print our molds.<br />
|'''Figure 4-b''' 'Screenshot' of the MakerWare software we used to print our molds.<br />
|'''Figure 4-c''' A roll of ABS filament used by the 3D-printer.<br />
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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).<br />
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{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_comb_and_box.jpg|200px]]<br />
|[[File:ETH Zurich 2014 small grid.JPG|200px]]<br />
|[[File:ETH Zurich diffusion plate.JPG|200px]]<br />
|[[File:ETH Zurich 2014 96 well all connected.jpeg|200px]]<br />
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|'''Figure 5-a''' Printed gel-comb and box. The box was autoclaved at 121 ⁰C. <br />
|'''Figure 5-b''' Printed millifluid grid with interconnected wells (edge length of 3 mm).<br />
|'''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).<br />
|'''Figure 5-d''' Printed 96-well plate, all wells (edge length of 5 mm) are connected.<br />
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<html><article id='Preparation'></html><br />
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==PDMS Chip Preparation==<br />
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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].<br />
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{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_first_mold_with_PDMS.jpg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion mold.JPG|200px]]<br />
|[[File:ETH Zurich 2014 final mold.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 final mold closeup.jpeg|200px]]<br />
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|'''Figure 6-a''' The very first mold design. PDMS stuck between the wells while removing it. <br />
|'''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).<br />
|'''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). <br />
|'''Figure 6-d''' Close up of the final mold design. The separate layers are clearly visible ('additive' manufacturing).<br />
|}<br />
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{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 broken chip.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 2 well diffusion chip upsidedown.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 PDMS diffusion chip final.jpeg|200px]]<br />
|[[File:ETH_Zurich_2014_final_chip_zoom.png|200px]]<br />
|-<br />
|'''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.<br />
|'''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).<br />
|'''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). <br />
|'''Figure 7-d''' Close up of the final PDMS chip. The channels are well defined and even small structures separated evenly from the mold.<br />
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<html><article id='movies'></html><br />
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==Time-Lapse Movies==<br />
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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.<br />
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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. <br />
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{|class="wikitable" style="background-color: white; text-align:center; width:100%; max-width: 650px; margin: auto;"<br />
|{{: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>}}<br />
|{{: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>}}<br />
|-<br />
|'''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.<br />
|'''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.<br />
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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>}}<br />
|{{: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>}}<br />
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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.<br />
|'''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.<br />
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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>}}<br />
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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.<br />
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{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/expresults/rrTeam:ETH Zurich/expresults/rr2014-10-18T03:21:19Z<p>Mbahls: /* Riboregulators */</p>
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<div>==Riboregulators==<br />
High basal expression from inactive inducible promoter (leakiness) is a common challenge in the implementation of robust biological control system. The problem is especially severe for systems that approximate digital Boolean logic or drive an amplified downstream response. In the first case an OFF state can be interpreted as ON resulting in incorrect computations. The second scenario leads to high levels of undesired final response. Many former iGEM teams have encountered unwanted basal expression of their genes of interest (''goi'') due to promoter leakiness ([https://2013.igem.org/Team:ETH_Zurich/Optimization ETH2013], [https://2012.igem.org/Team:Groningen/pigmentproduction Groningen2012], amongst others), resulting often in narrow optimal operation conditions for their biological devices. Since our system is based on Boolean Logic for its decisions and relies on downstream amplification to regenerate and propagate the signal as a first step we investigated strategies to improve tight gene control. Looking into the available literature we found riboregulators as a promising, highly generalizable approach to address the issue of leakiness<sup>[[Team:ETH_Zurich/project/references#refIsaacs|[32]]]</sup>. As a convenient proof of concept we coupled the system with [https://2014.igem.org/Team:ETH_Zurich/project/background#Quorum_Sensing quorum sensing modules] characterized the response and made the resulting parts available for the iGEM community in the [http://parts.igem.org/Main_Page Registry of Standard Biological Parts]. We suggest that this approach can be generalized to improve many of the inducible system contained in the registry<br />
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The riboregulator systems include two parts: 1) a ''cis''-repressed RBS in front of the ''goi'', and 2) a co-expressed ''trans''-activating RNA. <br />
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To characterize this system for reliable cell-to-cell communication, we combined the [https://2014.igem.org/Team:ETH_Zurich/data#Our_Favorite_New_Characterized_Parts riboregulator part] with promoters of the quorum-input-sensing systems used by our team ([https://2014.igem.org/Team:ETH_Zurich/data#Used_and_Characterized_Pre-Existing_Parts LuxI/LuxR, LasI/LasR, and RhlI/RhlR]). In our final [https://2014.igem.org/Team:ETH_Zurich/data#Gene_Circuit_and_Parts gene circuit], the riboregulators were intended for tight control of [https://2014.igem.org/Team:ETH_Zurich/project/background#Integrases integrases]. However, to have an easily quantifiable and proportional output, we initially used GFP as our riboregulated ''goi'' and measured the output fluorescence with a [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#Tecan_Infinite_M200_Pro.E2.84.A2 Tecan plate reader]. This approach is described below in the figure 1.<br />
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[[File:ETH Zurich2014 Riboregulator expresults.png|center|800px|thumb| '''Figure 2''' Characterization of the riboregulator system. First, a defined amount of the incoming signal molecule 3OC6-HSL binds to LuxR. After dimerization, the complex binds to its corresponding promoter pLux. Both, the ''trans''-activator(taR12y) and the ''cis''-repressor (crR12y) together with superfolder green fluorecent protein (sfGFP), are under control of pLux. Upon induction with LuxR/3OC6-HSL, both are transcribed simultaneously and the taR12y (key symbol) opens up the crR12y sequence (lock) in front of the sfGFP mRNA. This enables translation and sfGFP production (left-hand side). Low transcription (leakiness) gives only small concentrations of mRNA, as a result 'lock' (''cis''-repressor) and 'key' (''trans''-activator) structures do not encounter each other and the translation is not enabled (right-hand side).]]<br />
The first set of experiments was conducted with cells transformed with two different pPaB plasmids. These plasmids contained either the pBR322 origin (pMB1) or the p15A origin, yielding a stable two-plasmid system with about 15-20 copies per plasmid and cell (medium-copy). Furthermore the published riboregulator sequence includes two forbidden restriction sites (EcoRI and XbaI), since the sites removal could affect the folding or functionality of the part, the original sequences were used as a control. After characterizing the original part the restriction sites were removed by two different approaches: a) multiple site-directed mutagenesis, b) blunting and ligation (Klenow and T4 DNA polymerase). The new Biobrick compatible riboregulator was tested to confirm that no severe loss-of-function occurred due to the site-removal.<br />
In a third step, the construct was transferred into the [http://parts.igem.org/Part:pSB1C3 pSB1C3] backbone (pMB1 origin, high copy number of 100-300) to be in line with the [http://parts.igem.org/Help:Standards/Assembly BioBrick Standard]. Below, you can find the corresponding graphs in figure 2 and figure3, respectively. Future characterization of these constructs should allow us to explore the leakiness dependence on plasmid amount and if the tight expression is robust for different copy numbers. <br />
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We have characterized promoters response of the three quorum-sensing systems. These are [https://2014.igem.org/Team:ETH_Zurich/data#Used_and_Characterized_Pre-Existing_Parts pLux, pLas, and pRhl]. We first measured the transfer functions with a non-regulated RBS in front of sfGFP, i. e. the amount of fluorescence in dependence of the inducer concentration present and compared the response with the corresponding riboregulated constructs with and without the forbidden restriction sites. All experiments were carried out in 96-well [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#Tecan_Infinite_M200_Pro.E2.84.A2 microtiter plate format] for 10 h. This kinetic data-sets were also used for parameter-fitting in our [https://2014.igem.org/Team:ETH_Zurich/modeling/qs#Retrieving_degradation_rates modeling approach].<br />
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We found an about 60-fold reduced basal GFP expression and an increased signal-to-noise ratio of about 6-fold when using our riboregulator construct, as compared to a non-regulated [http://parts.igem.org/Part:BBa_B0034 reference RBS (B0034)]. These results are summarized in figure 2. The BioBrick conform module was confirmed to show no loss-of-funtion due to EcoRI and XbaI site removal. Interestingly the sensitivity towards the inducer was reduced (see figure 3). Is interesting to speculate if small changes in the sequences flanking the riboregulator could be used as a way to switch the response curve.<br />
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{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 with regulator.png|center|600px]] <br />
|[[File:ETH Zurich 2014 without sites.png|center|600px]]<br />
|-<br />
|'''Figure 3''' Improved signal-to-noise ratio and decreased basal GFP expression (leakiness) due to the use of a riboregulator in combination with a quorum-sensing module. The fluorescence per OD<sub>600</sub> is shown for the LuxR-system with a complete riboregulator over an inducer-range of 10<sup>-13</sup> M to 10<sup>-5</sup> M (dashed, light blue). An incomplete riboregulator without the ''trans''-activator shows the expected reduced sensitivity towards the inducer (dark blue). As a reference, a system with a [http://parts.igem.org/Part:BBa_B0034 non-regulated RBS (BBa_B0034)] is shown (light blue). Data points are mean values of triplicate measurements in 96-well microtiter plates 200 min after induction &plusmn; standard deviation. For the full data set and kinetics please [https://2014.igem.org/Team:ETH_Zurich/contact contact] us or visit the [https://2014.igem.org/Team:ETH_Zurich/data/raw raw data] page.<br />
|'''Figure 4''' Confirmation of the improved signal-to-noise ratio and decreased basal GFP expression (leakiness) due to the use of a riboregulator without (w/o) EcoRI and XbaI restriction sites in combination with a quorum-sensing module. The fluorescence per OD<sub>600</sub> is shown for the LuxR-system with an unchanged riboregulator (dashed, light blue) and a regulator with a changed sequence due to EcoRI and XbaI restriction site removal (dashed, dark blue). The inducer range covers 10<sup>-13</sup> M to 10<sup>-5</sup> M. As a reference, a system with a [http://parts.igem.org/Part:BBa_B0034 non-regulated RBS (BBa_B0034)] is shown (light blue). Data points are mean values of triplicate measurements in 96-well microtiter plates 200 min after induction &plusmn; standard deviation. For the full data set and kinetics please [https://2014.igem.org/Team:ETH_Zurich/contact contact] us or visit the [https://2014.igem.org/Team:ETH_Zurich/data/raw raw data] page.<br />
|}<br />
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{{:Team:ETH_Zurich/tpl/topbutton|blue}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/expresults/rrTeam:ETH Zurich/expresults/rr2014-10-18T03:20:52Z<p>Mbahls: /* Riboregulators */</p>
<hr />
<div>==Riboregulators==<br />
High basal expression from inactive inducible promoter (leakiness) is a common challenge in the implementation of robust biological control system. The problem is especially severe for systems that approximate digital Boolean logic or drive an amplified downstream response. In the first case an OFF state can be interpreted as ON resulting in incorrect computations. The second scenario leads to high levels of undesired final response. Many former iGEM teams have encountered unwanted basal expression of their genes of interest (''goi'') due to promoter leakiness ([https://2013.igem.org/Team:ETH_Zurich/Optimization ETH2013], [https://2012.igem.org/Team:Groningen/pigmentproduction Groningen2012], amongst others), resulting often in narrow optimal operation conditions for their biological devices. Since our system is based on Boolean Logic for its decisions and relies on downstream amplification to regenerate and propagate the signal as a first step we investigated strategies to improve tight gene control. Looking into the available literature we found riboregulators as a promising, highly generalizable approach to address the issue of leakiness<sup>[[Team:ETH_Zurich/project/references#refIsaacs|[32]]]</sup>. As a convenient proof of concept we coupled the system with [https://2014.igem.org/Team:ETH_Zurich/project/background#Quorum_Sensing quorum sensing modules] characterized the response and made the resulting parts available for the iGEM community in the [http://parts.igem.org/Main_Page Registry of Standard Biological Parts]. We suggest that this approach can be generalized to improve many of the inducible system contained in the registry<br />
<br />
The riboregulator systems include two parts: 1) a ''cis''-repressed RBS in front of the ''goi'', and 2) a co-expressed ''trans''-activating RNA. <br />
<br />
<br />
To characterize this system for reliable cell-to-cell communication, we combined the [https://2014.igem.org/Team:ETH_Zurich/data#Our_Favorite_New_Characterized_Parts riboregulator part] with promoters of the quorum-input-sensing systems used by our team ([https://2014.igem.org/Team:ETH_Zurich/data#Used_and_Characterized_Pre-Existing_Parts LuxI/LuxR, LasI/LasR, and RhlI/RhlR]). In our final [https://2014.igem.org/Team:ETH_Zurich/data#Gene_Circuit_and_Parts gene circuit], the riboregulators were intended for tight control of [https://2014.igem.org/Team:ETH_Zurich/project/background#Integrases integrases]. However, to have an easily quantifiable and proportional output, we initially used GFP as our riboregulated ''goi'' and measured the output fluorescence with a [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#Tecan_Infinite_M200_Pro.E2.84.A2 Tecan plate reader]. This approach is described below in the figure 1.<br />
<br />
<br />
[[File:ETH Zurich2014 Riboregulator expresults.png|center|800px|thumb| '''Figure 2''' Characterization of the riboregulator system. First, a defined amount of the incoming signal molecule 3OC6-HSL binds to LuxR. After dimerization, the complex binds to its corresponding promoter pLux. Both, the ''trans''-activator(taR12y) and the ''cis''-repressor (crR12y) together with superfolder green fluorecent protein (sfGFP), are under control of pLux. Upon induction with LuxR/3OC6-HSL, both are transcribed simultaneously and the taR12y (key symbol) opens up the crR12y sequence (lock) in front of the sfGFP mRNA. This enables translation and sfGFP production (left-hand side). Low transcription (leakiness) gives only small concentrations of mRNA, as a result 'lock' (''cis''-repressor) and 'key' (''trans''-activator) structures do not encounter each other and the translation is not enabled (right-hand side).]]<br />
The first set of experiments was conducted with cells transformed with two different pPaB plasmids. These plasmids contained either the pBR322 origin (pMB1) or the p15A origin, yielding a stable two-plasmid system with about 15-20 copies per plasmid and cell (medium-copy). Furthermore the published riboregulator sequence includes two forbidden restriction sites (EcoRI and XbaI), since the sites removal could affect the folding or functionality of the part, the original sequences were used as a control. After characterizing the original part the restriction sites were removed by two different approaches: a) multiple site-directed mutagenesis, b) blunting and ligation (Klenow and T4 DNA polymerase). The new Biobrick compatible riboregulator was tested to confirm that no severe loss-of-function occurred due to the site-removal.<br />
In a third step, the construct was transferred into the [http://parts.igem.org/Part:pSB1C3 pSB1C3] backbone (pMB1 origin, high copy number of 100-300) to be in line with the [http://parts.igem.org/Help:Standards/Assembly BioBrick Standard]. Below, you can find the corresponding graphs in figure 2 and figure3, respectively. Future characterization of these constructs should allow us to explore the leakiness dependence on plasmid amount and if the tight expression is robust for different copy numbers. <br />
<br />
We have characterized promoters response of the three quorum-sensing systems. These are [https://2014.igem.org/Team:ETH_Zurich/data#Used_and_Characterized_Pre-Existing_Parts pLux, pLas, and pRhl]. We first measured the transfer functions with a non-regulated RBS in front of sfGFP, i. e. the amount of fluorescence in dependence of the inducer concentration present and compared the response with the corresponding riboregulated constructs with and without the forbidden restriction sites. All experiments were carried out in 96-well [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#Tecan_Infinite_M200_Pro.E2.84.A2 microtiter plate format] for 10 h. This kinetic data-sets were also used for parameter-fitting in our [https://2014.igem.org/Team:ETH_Zurich/modeling/qs#Retrieving_degradation_rates modeling approach].<br />
<br />
We found an about 60-fold reduced basal GFP expression and an increased signal-to-noise ratio of about 6-fold when using our riboregulator construct, as compared to a non-regulated [http://parts.igem.org/Part:BBa_B0034 reference RBS (B0034)]. These results are summarized in figure 2. The BioBrick conform module was confirmed to show no loss-of-funtion due to EcoRI and XbaI site removal. Interestingly the sensitivity towards the inducer was reduced (see figure 3). Is interesting to speculate if small changes in the sequences flanking the riboregulator could be used as a way to switch the response curve.<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 with regulator.png|center|600px]] <br />
|[[File:ETH Zurich 2014 without sites.png|center|600px]]<br />
|-<br />
|'''Figure 3''' Improved signal-to-noise ratio and decreased basal GFP expression (leakiness) due to the use of a riboregulator in combination with a quorum-sensing module. The fluorescence per OD<sub>600</sub> is shown for the LuxR-system with a complete riboregulator over an inducer-range of 10<sup>-13</sup> M to 10<sup>-5</sup> M (dashed, light blue). An incomplete riboregulator without the ''trans''-activator shows the expected reduced sensitivity towards the inducer (dark blue). As a reference, a system with a [http://parts.igem.org/Part:BBa_B0034 non-regulated RBS (BBa_B0034)] is shown (light blue). Data points are mean values of triplicate measurements in 96-well microtiter plates 200 min after induction &plusmn; standard deviation. For the full data set and kinetics please [https://2014.igem.org/Team:ETH_Zurich/contact contact] us or visit the [https://2014.igem.org/Team:ETH_Zurich/data/raw raw data] page.<br />
|'''Figure 3''' Confirmation of the improved signal-to-noise ratio and decreased basal GFP expression (leakiness) due to the use of a riboregulator without (w/o) EcoRI and XbaI restriction sites in combination with a quorum-sensing module. The fluorescence per OD<sub>600</sub> is shown for the LuxR-system with an unchanged riboregulator (dashed, light blue) and a regulator with a changed sequence due to EcoRI and XbaI restriction site removal (dashed, dark blue). The inducer range covers 10<sup>-13</sup> M to 10<sup>-5</sup> M. As a reference, a system with a [http://parts.igem.org/Part:BBa_B0034 non-regulated RBS (BBa_B0034)] is shown (light blue). Data points are mean values of triplicate measurements in 96-well microtiter plates 200 min after induction &plusmn; standard deviation. For the full data set and kinetics please [https://2014.igem.org/Team:ETH_Zurich/contact contact] us or visit the [https://2014.igem.org/Team:ETH_Zurich/data/raw raw data] page.<br />
|}<br />
<br />
<br />
{{:Team:ETH_Zurich/tpl/topbutton|blue}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/expresults/qsTeam:ETH Zurich/expresults/qs2014-10-18T03:19:21Z<p>Mbahls: /* Quorum Sensing */</p>
<hr />
<div><br/><br />
== Quorum Sensing ==<br />
<br />
For our Mosia''coli'' project, we were looking for molecular systems that allow orthogonal [https://2014.igem.org/Team:ETH_Zurich/project/background#Quorum_Sensing cell-to-cell communication] in order to implement connected [https://2014.igem.org/Team:ETH_Zurich/modeling/xor#XOR_Logic_Gate XOR logic gates]. We decided to exploit the quorum sensing systems [https://2014.igem.org/Team:ETH_Zurich/data#Gene_Circuit LuxI/LuxR, LasI/LasR, and RhlI/RhlR] in order to achieve the required orthogonal cell-to-cell communication. We developed a [https://2014.igem.org/Team:ETH_Zurich/modeling/qs model] for these cellular information processing. Even though the corresponding inducer molecules are commercially available and the systems often used, in particular in iGEM projects (e.g. [http://parts.igem.org/Part:BBa_R0062 pLux (BBa_R0062)], '[http://parts.igem.org/Frequently_Used_Parts Top 10 Most used promoters]' with 246 uses), potential crosstalk activity between the different systems may be a severe problem (e. g. [https://2013.igem.org/Team:Tokyo_Tech/Project/Ninja_State_Switching Tokyo_Tech 2013], [https://2011.igem.org/Team:Peking_S/project/wire/matrix Peking University 2011]).<br />
<br />
<br />
In order to address this challenge, we measured a) a given promoter with its corresponding regulator and a different inducer molecule, b) a given promoter with an unspecific regulator and a particular inducer, c) a given promoter with both regulator and inducer being unspecific, and always included the correct combination of inducer molecule, regulator and promoter as a positive control. This gives in total 27 possible combinations. The output was assessed via sfGFP and measured in terms of fluorescence on microtiter-plate scale.<br />
[[File:ETH Zurich Crosstalk.png|1500px|center|thumb|'''Figure 1''' Each quorum sensing system is based on three components: a signaling molecule, a regulatory protein and a promoter. These elements are here ordered into three layers. Cross-talk evaluation can be done by comparing all combinations of those three elements. After collecting the [https://2014.igem.org/Team:ETH_Zurich/expresults experimental data] of all possible pathways, we modeled their influence.]]<br />
<br/><br />
===Summary of experimental results regarding quorum sensing===<br />
The following matrices serve as an overview summarizing the most significant results of our experiments to characterize crosstalk on different levels. <br />
On the horizontal top row we see the three different inducer molecules (3OC12-HSL, 3OC6-HSL, C4-HSL). In the top left corner we see the quorum sensing promoter used for all the experiments summarized in this matrix. On the vertical axis we see the three regulators ( [https://2014.igem.org/Team:ETH_Zurich/data#Used_and_Characterized_Pre-Existing_Parts LuxR, LasR, RhlR]). <br />
These matrices are giving an overview of the experimental results conducted in relation with [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing quorum sensing] and crosstalk. The graph shown in each matrix on the very top left describes the situation where the correct autoinducer molecule has bound the corresponding regulator and this complex has then induced the correct promoter. <br />
The solid lines in the graphs show the [https://2014.igem.org/Team:ETH_Zurich/modeling/qs model data], whereas the data points indicated with standard deviation show experimental data in triplicates (mean values of triplicate micro titerplate measurements). <br />
<br />
<br />
{{:Team:ETH_Zurich/expresults/qs/tab-plux}}<br />
<br />
<br />
{{:Team:ETH_Zurich/expresults/qs/tab-plas}}<br />
<br />
<br />
{{:Team:ETH_Zurich/expresults/qs/tab-prhl}}<br />
<br />
===Conclusion of crosstalk experiments===<br />
<br />
As shown in the graphs in the matrices above, we found and quantitatively characterized all three levels of crosstalk. The three levels were the following:<br />
*A given promoter with its corresponding regulator and a different inducer molecule<br />
*A given promoter with an unspecific regulator and a particular inducer<br />
*A given promoter with both regulator and inducer being unspecific<br />
Unspecific inducers binding to the regulators as well as unspecific binding of the regulator to another promoter species was observed in almost all possible combinations. <br />
To conclude, we were not able to find an orthogonal quorum sensing pair out of the three systems investigated ([https://2014.igem.org/Team:ETH_Zurich/data#Used_and_Characterized_Pre-Existing_Parts LuxI/LuxR, LasI/LasR, or RhlI/RhlR]). While we see a significant effect when implementing the influence of these crosstalks (on an inducer-, regulator- and promoter-level) in our [https://2014.igem.org/Team:ETH_Zurich/modeling/whole whole cell model], the logic gate still continues to function for a range of inputs at physiological concentrations.<br />
<br />
<br />
<br />
{{:Team:ETH_Zurich/tpl/topbutton|red}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/project/infoproTeam:ETH Zurich/project/infopro2014-10-18T03:03:48Z<p>Mbahls: /* Why we chose this track */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Information Processing}}<br />
<html><article></html><br />
<br />
== Information Processing ==<br />
<br />
<br />
=== About the track ===<br />
<br />
<br />
[https://2014.igem.org/Tracks/Information_Processing ''«Information Processing in iGEM covers a diverse range of projects. IP teams are not trying to solve a real world problem with practical applications, but to tackle an interesting problem that might otherwise not attract attention. Teams enter this track if they are attempting projects such as building elements of a biological computer, creating a game using biology or working on a signal processing challenges. Engineering ways to make biological systems perform computation is one of the core goals of synthetic biology. We generally work at the DNA level, engineering systems to function using BioBricks. In most biological systems, protein-protein interactions are where the majority of processing takes place. Being able to design proteins to accomplish computation would allow for systems to function on a much faster timescale than the current transcription-translation paradigm»'' (iGEM mainpage)]<br />
<br />
=== Why we chose this track ===<br />
<br />
With our project, Mosai''coli'', we investigate the emergence of complex patterns from simple mathematical rules. Such rules can be reduced to Boolean logic gates, in our case XOR gates. The computations were implemented with integrases, proteins modifying DNA between specific sites. These modifications in turn influence the expression of other proteins which can then indicate the previous change on the genetic level. Additionally chemical wiring of the information to the next cells happens, this again on the protein or small-molecule level. This wiring of information and repeated information processing allows the construction of cellular automata and eventually biocomputers using the analogy of electrical circuits.<br />
<br />
=== The goal: Emergence of patterns via information processing===<br />
We implement a cellular automaton with bacterial cells containing our logic circuitry. Each bacterial colony serves as a core, computing an XOR gate. First, a sensor device detects the input, ''N''-acyl homoserine lactones (AHL). Then, the cell integrates this signal through a logic gate, performed by serine integrases by sensing on the protein level and acting on the DNA level. A necessary post-processing step allows then the production of a new AHL variant due to activated gene expression through the integrase. Meanwhile, green fluorescent protein (GFP) indicates the state of the colony and serves as a long-lasting visual read out. The produced AHL output-signal then propagates in a directed fashion through a millifluidic grid to the next bacterial colony. This iterative process faces the challenges of leakiness, cross-talk, protein-level computation and exact diffusion steps. This information pathway is shown in figure 1.<br />
<br />
<br />
[[File:ETH_Zurich2014_info_processing1.png|center|800px|thumb|'''Figure 1 The information pathway in the project Mosai''coli'' on a colony level.''' Each cell colony is part of a greater memory unit (left-hand side) and gets its input information from the neighboring colonies. The input is given in the form of two different AHLs, here named A and B. This information is then step-wise processed in each of the colonies: sensing - computing - producing - sending. The output molecules serve then as the input for the next colony (middle and left-hand side). This iterative process allows the information processing from the top row of the memory unit to the bottom by chemical wiring via diffusing signals and bio-computations inside of the colonies.]]<br />
<br />
<br />
<br />
=== The steps involved: From sensing to sending===<br />
<br />
=== Sensing ===<br />
<br />
Diffused AHL molecules are identified via the [https://2014.igem.org/Team:ETH_Zurich/expresults#Quorum_Sensing quorum sensing machinery]. The [https://2014.igem.org/Team:ETH_Zurich/modeling/qs transfer function] between the sensed AHL concentration and the promoter activation deviates usually from the ideal one due to leakiness and crosstalk between different types of AHL molecules, regulatory proteins and promoters (we [https://2014.igem.org/Team:ETH_Zurich/expresults/qs#Quorum_Sensing characterized] it for [http://parts.igem.org/Part:BBa_R0062 Lux promoter (BBa_R0062)], [http://parts.igem.org/Part:BBa_R0079 Las promoter (BBa_R0079)] and [http://parts.igem.org/Part:BBa_R0071 Rhl promoter (BBa_R0071)]). To limit the risk of error propagation, we implemented a [http://parts.igem.org/Part:BBa_K1541000 riboregulated system].<br />
<br />
=== Computing ===<br />
<br />
[https://2014.igem.org/Team:ETH_Zurich/expresults#Integrases Integrases] compute an [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR logic gate] via single switching or double switching of a terminator. Their behavior depends on a set of [https://2014.igem.org/Team:ETH_Zurich/modeling/int parameters].<br />
<br />
=== Producing ===<br />
<br />
To propagate the computational results, the output has to be translated again into a quorum sensing molecule. Therefore a synthase is expressed to catalyze the production of a new AHL signal.<br />
<br />
=== Memorising ===<br />
<br />
Alongside the AHL synthase, GFP is produced according to the XOR truth table. The fluorescent protein acts both as long term memory for the colonies in ON-state and as visual signal, storing and showing the propagating pattern formation.<br />
<br />
=== Sending ===<br />
<br />
The produced AHL diffuses through channels on the memory unit to the rows below, becoming the input of the following information processing layer. The speed of this 'wiring' by directed diffusion determines the time needed for the pattern to emerge (as long as the channels are relatively long and diffusion is the time-limting step as compared to the actual computation itself). You can find out more information about diffusion and chip design on our [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel modeling page], our [https://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion_On_Chip experimental results page] and our [https://2014.igem.org/Team:ETH_Zurich/lab/chip chip page].<br />
<br />
<br />
The overall process is summarized for the chip, or memory unit, level in figure 2.<br />
<br />
<br />
[[File:ETH_Zurich_Information_Processing_chip.png|center|600px|thumb|'''Figure 2 The information pathway in the project Mosai''coli'' on the chip level.''' After initializing the signal in the top row, it propagates through the wells chemically wired by directed diffusion. In each well, a bacterial colony has to be able to proceed in the information pathway: sensing, integrating (computing), producing and sending. These successive iterations leads to possible error propagation and robustness is one major issue of our system.]]<br />
<br />
<br />
<html></article></html><br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/project/infoproTeam:ETH Zurich/project/infopro2014-10-18T02:57:32Z<p>Mbahls: /* Why we chose this track */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Information Processing}}<br />
<html><article></html><br />
<br />
== Information Processing ==<br />
<br />
<br />
=== About the track ===<br />
<br />
<br />
[https://2014.igem.org/Tracks/Information_Processing ''«Information Processing in iGEM covers a diverse range of projects. IP teams are not trying to solve a real world problem with practical applications, but to tackle an interesting problem that might otherwise not attract attention. Teams enter this track if they are attempting projects such as building elements of a biological computer, creating a game using biology or working on a signal processing challenges. Engineering ways to make biological systems perform computation is one of the core goals of synthetic biology. We generally work at the DNA level, engineering systems to function using BioBricks. In most biological systems, protein-protein interactions are where the majority of processing takes place. Being able to design proteins to accomplish computation would allow for systems to function on a much faster timescale than the current transcription-translation paradigm»'' (iGEM mainpage)]<br />
<br />
=== Why we chose this track ===<br />
<br />
With our project, Mosai''coli'', we investigate the emergence of complex patterns from simple mathematical rules. Such rules can be reduced to Boolean logic gates, in our case XOR gates. The computations were implemented with integrases, proteins modifying DNA between specific sites. These modifications in turn influence the expression of other proteins which can then indicate the previous change on the genetic level and chemically wire the information to the next cells, again on the protein or small-molecule level. This wiring of information and repeated information processing allows the construction of cellular automata and eventually biocomputers using the analogy of electrical circuits.<br />
<br />
=== The goal: Emergence of patterns via information processing===<br />
We implement a cellular automaton with bacterial cells containing our logic circuitry. Each bacterial colony serves as a core, computing an XOR gate. First, a sensor device detects the input, ''N''-acyl homoserine lactones (AHL). Then, the cell integrates this signal through a logic gate, performed by serine integrases by sensing on the protein level and acting on the DNA level. A necessary post-processing step allows then the production of a new AHL variant due to activated gene expression through the integrase. Meanwhile, green fluorescent protein (GFP) indicates the state of the colony and serves as a long-lasting visual read out. The produced AHL output-signal then propagates in a directed fashion through a millifluidic grid to the next bacterial colony. This iterative process faces the challenges of leakiness, cross-talk, protein-level computation and exact diffusion steps. This information pathway is shown in figure 1.<br />
<br />
<br />
[[File:ETH_Zurich2014_info_processing1.png|center|800px|thumb|'''Figure 1 The information pathway in the project Mosai''coli'' on a colony level.''' Each cell colony is part of a greater memory unit (left-hand side) and gets its input information from the neighboring colonies. The input is given in the form of two different AHLs, here named A and B. This information is then step-wise processed in each of the colonies: sensing - computing - producing - sending. The output molecules serve then as the input for the next colony (middle and left-hand side). This iterative process allows the information processing from the top row of the memory unit to the bottom by chemical wiring via diffusing signals and bio-computations inside of the colonies.]]<br />
<br />
<br />
<br />
=== The steps involved: From sensing to sending===<br />
<br />
=== Sensing ===<br />
<br />
Diffused AHL molecules are identified via the [https://2014.igem.org/Team:ETH_Zurich/expresults#Quorum_Sensing quorum sensing machinery]. The [https://2014.igem.org/Team:ETH_Zurich/modeling/qs transfer function] between the sensed AHL concentration and the promoter activation deviates usually from the ideal one due to leakiness and crosstalk between different types of AHL molecules, regulatory proteins and promoters (we [https://2014.igem.org/Team:ETH_Zurich/expresults/qs#Quorum_Sensing characterized] it for [http://parts.igem.org/Part:BBa_R0062 Lux promoter (BBa_R0062)], [http://parts.igem.org/Part:BBa_R0079 Las promoter (BBa_R0079)] and [http://parts.igem.org/Part:BBa_R0071 Rhl promoter (BBa_R0071)]). To limit the risk of error propagation, we implemented a [http://parts.igem.org/Part:BBa_K1541000 riboregulated system].<br />
<br />
=== Computing ===<br />
<br />
[https://2014.igem.org/Team:ETH_Zurich/expresults#Integrases Integrases] compute an [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR logic gate] via single switching or double switching of a terminator. Their behavior depends on a set of [https://2014.igem.org/Team:ETH_Zurich/modeling/int parameters].<br />
<br />
=== Producing ===<br />
<br />
To propagate the computational results, the output has to be translated again into a quorum sensing molecule. Therefore a synthase is expressed to catalyze the production of a new AHL signal.<br />
<br />
=== Memorising ===<br />
<br />
Alongside the AHL synthase, GFP is produced according to the XOR truth table. The fluorescent protein acts both as long term memory for the colonies in ON-state and as visual signal, storing and showing the propagating pattern formation.<br />
<br />
=== Sending ===<br />
<br />
The produced AHL diffuses through channels on the memory unit to the rows below, becoming the input of the following information processing layer. The speed of this 'wiring' by directed diffusion determines the time needed for the pattern to emerge (as long as the channels are relatively long and diffusion is the time-limting step as compared to the actual computation itself). You can find out more information about diffusion and chip design on our [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel modeling page], our [https://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion_On_Chip experimental results page] and our [https://2014.igem.org/Team:ETH_Zurich/lab/chip chip page].<br />
<br />
<br />
The overall process is summarized for the chip, or memory unit, level in figure 2.<br />
<br />
<br />
[[File:ETH_Zurich_Information_Processing_chip.png|center|600px|thumb|'''Figure 2 The information pathway in the project Mosai''coli'' on the chip level.''' After initializing the signal in the top row, it propagates through the wells chemically wired by directed diffusion. In each well, a bacterial colony has to be able to proceed in the information pathway: sensing, integrating (computing), producing and sending. These successive iterations leads to possible error propagation and robustness is one major issue of our system.]]<br />
<br />
<br />
<html></article></html><br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/project/infoproTeam:ETH Zurich/project/infopro2014-10-18T02:52:45Z<p>Mbahls: /* Sending */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Information Processing}}<br />
<html><article></html><br />
<br />
== Information Processing ==<br />
<br />
<br />
=== About the track ===<br />
<br />
<br />
[https://2014.igem.org/Tracks/Information_Processing ''«Information Processing in iGEM covers a diverse range of projects. IP teams are not trying to solve a real world problem with practical applications, but to tackle an interesting problem that might otherwise not attract attention. Teams enter this track if they are attempting projects such as building elements of a biological computer, creating a game using biology or working on a signal processing challenges. Engineering ways to make biological systems perform computation is one of the core goals of synthetic biology. We generally work at the DNA level, engineering systems to function using BioBricks. In most biological systems, protein-protein interactions are where the majority of processing takes place. Being able to design proteins to accomplish computation would allow for systems to function on a much faster timescale than the current transcription-translation paradigm»'' (iGEM mainpage)]<br />
<br />
=== Why we chose this track ===<br />
<br />
With our project, Mosai''coli'', we investigate the emergence of complex patterns from simple mathematical rules. Such rules can be reduced to Boolean logic gates, in our case XOR gates. The computations were implemented with integrases, proteins modifing DNA between specific sites. These modifications in turn influence the expression of other proteins which can then indicate the previous change on the genetic level and chemically wire the information to the next cells, again on the protein or small-molecule level. This wiring of information and repeated information processing allows the construction of cellular automata and eventually biocomputers.<br />
<br />
=== The goal: Emergence of patterns via information processing===<br />
We implement a cellular automaton with bacterial cells containing our logic circuitry. Each bacterial colony serves as a core, computing an XOR gate. First, a sensor device detects the input, ''N''-acyl homoserine lactones (AHL). Then, the cell integrates this signal through a logic gate, performed by serine integrases by sensing on the protein level and acting on the DNA level. A necessary post-processing step allows then the production of a new AHL variant due to activated gene expression through the integrase. Meanwhile, green fluorescent protein (GFP) indicates the state of the colony and serves as a long-lasting visual read out. The produced AHL output-signal then propagates in a directed fashion through a millifluidic grid to the next bacterial colony. This iterative process faces the challenges of leakiness, cross-talk, protein-level computation and exact diffusion steps. This information pathway is shown in figure 1.<br />
<br />
<br />
[[File:ETH_Zurich2014_info_processing1.png|center|800px|thumb|'''Figure 1 The information pathway in the project Mosai''coli'' on a colony level.''' Each cell colony is part of a greater memory unit (left-hand side) and gets its input information from the neighboring colonies. The input is given in the form of two different AHLs, here named A and B. This information is then step-wise processed in each of the colonies: sensing - computing - producing - sending. The output molecules serve then as the input for the next colony (middle and left-hand side). This iterative process allows the information processing from the top row of the memory unit to the bottom by chemical wiring via diffusing signals and bio-computations inside of the colonies.]]<br />
<br />
<br />
<br />
=== The steps involved: From sensing to sending===<br />
<br />
=== Sensing ===<br />
<br />
Diffused AHL molecules are identified via the [https://2014.igem.org/Team:ETH_Zurich/expresults#Quorum_Sensing quorum sensing machinery]. The [https://2014.igem.org/Team:ETH_Zurich/modeling/qs transfer function] between the sensed AHL concentration and the promoter activation deviates usually from the ideal one due to leakiness and crosstalk between different types of AHL molecules, regulatory proteins and promoters (we [https://2014.igem.org/Team:ETH_Zurich/expresults/qs#Quorum_Sensing characterized] it for [http://parts.igem.org/Part:BBa_R0062 Lux promoter (BBa_R0062)], [http://parts.igem.org/Part:BBa_R0079 Las promoter (BBa_R0079)] and [http://parts.igem.org/Part:BBa_R0071 Rhl promoter (BBa_R0071)]). To limit the risk of error propagation, we implemented a [http://parts.igem.org/Part:BBa_K1541000 riboregulated system].<br />
<br />
=== Computing ===<br />
<br />
[https://2014.igem.org/Team:ETH_Zurich/expresults#Integrases Integrases] compute an [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR logic gate] via single switching or double switching of a terminator. Their behavior depends on a set of [https://2014.igem.org/Team:ETH_Zurich/modeling/int parameters].<br />
<br />
=== Producing ===<br />
<br />
To propagate the computational results, the output has to be translated again into a quorum sensing molecule. Therefore a synthase is expressed to catalyze the production of a new AHL signal.<br />
<br />
=== Memorising ===<br />
<br />
Alongside the AHL synthase, GFP is produced according to the XOR truth table. The fluorescent protein acts both as long term memory for the colonies in ON-state and as visual signal, storing and showing the propagating pattern formation.<br />
<br />
=== Sending ===<br />
<br />
The produced AHL diffuses through channels on the memory unit to the rows below, becoming the input of the following information processing layer. The speed of this 'wiring' by directed diffusion determines the time needed for the pattern to emerge (as long as the channels are relatively long and diffusion is the time-limting step as compared to the actual computation itself). You can find out more information about diffusion and chip design on our [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel modeling page], our [https://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion_On_Chip experimental results page] and our [https://2014.igem.org/Team:ETH_Zurich/lab/chip chip page].<br />
<br />
<br />
The overall process is summarized for the chip, or memory unit, level in figure 2.<br />
<br />
<br />
[[File:ETH_Zurich_Information_Processing_chip.png|center|600px|thumb|'''Figure 2 The information pathway in the project Mosai''coli'' on the chip level.''' After initializing the signal in the top row, it propagates through the wells chemically wired by directed diffusion. In each well, a bacterial colony has to be able to proceed in the information pathway: sensing, integrating (computing), producing and sending. These successive iterations leads to possible error propagation and robustness is one major issue of our system.]]<br />
<br />
<br />
<html></article></html><br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/project/infoproTeam:ETH Zurich/project/infopro2014-10-18T02:50:29Z<p>Mbahls: /* Sending */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Information Processing}}<br />
<html><article></html><br />
<br />
== Information Processing ==<br />
<br />
<br />
=== About the track ===<br />
<br />
<br />
[https://2014.igem.org/Tracks/Information_Processing ''«Information Processing in iGEM covers a diverse range of projects. IP teams are not trying to solve a real world problem with practical applications, but to tackle an interesting problem that might otherwise not attract attention. Teams enter this track if they are attempting projects such as building elements of a biological computer, creating a game using biology or working on a signal processing challenges. Engineering ways to make biological systems perform computation is one of the core goals of synthetic biology. We generally work at the DNA level, engineering systems to function using BioBricks. In most biological systems, protein-protein interactions are where the majority of processing takes place. Being able to design proteins to accomplish computation would allow for systems to function on a much faster timescale than the current transcription-translation paradigm»'' (iGEM mainpage)]<br />
<br />
=== Why we chose this track ===<br />
<br />
With our project, Mosai''coli'', we investigate the emergence of complex patterns from simple mathematical rules. Such rules can be reduced to Boolean logic gates, in our case XOR gates. The computations were implemented with integrases, proteins modifing DNA between specific sites. These modifications in turn influence the expression of other proteins which can then indicate the previous change on the genetic level and chemically wire the information to the next cells, again on the protein or small-molecule level. This wiring of information and repeated information processing allows the construction of cellular automata and eventually biocomputers.<br />
<br />
=== The goal: Emergence of patterns via information processing===<br />
We implement a cellular automaton with bacterial cells containing our logic circuitry. Each bacterial colony serves as a core, computing an XOR gate. First, a sensor device detects the input, ''N''-acyl homoserine lactones (AHL). Then, the cell integrates this signal through a logic gate, performed by serine integrases by sensing on the protein level and acting on the DNA level. A necessary post-processing step allows then the production of a new AHL variant due to activated gene expression through the integrase. Meanwhile, green fluorescent protein (GFP) indicates the state of the colony and serves as a long-lasting visual read out. The produced AHL output-signal then propagates in a directed fashion through a millifluidic grid to the next bacterial colony. This iterative process faces the challenges of leakiness, cross-talk, protein-level computation and exact diffusion steps. This information pathway is shown in figure 1.<br />
<br />
<br />
[[File:ETH_Zurich2014_info_processing1.png|center|800px|thumb|'''Figure 1 The information pathway in the project Mosai''coli'' on a colony level.''' Each cell colony is part of a greater memory unit (left-hand side) and gets its input information from the neighboring colonies. The input is given in the form of two different AHLs, here named A and B. This information is then step-wise processed in each of the colonies: sensing - computing - producing - sending. The output molecules serve then as the input for the next colony (middle and left-hand side). This iterative process allows the information processing from the top row of the memory unit to the bottom by chemical wiring via diffusing signals and bio-computations inside of the colonies.]]<br />
<br />
<br />
<br />
=== The steps involved: From sensing to sending===<br />
<br />
=== Sensing ===<br />
<br />
Diffused AHL molecules are identified via the [https://2014.igem.org/Team:ETH_Zurich/expresults#Quorum_Sensing quorum sensing machinery]. The [https://2014.igem.org/Team:ETH_Zurich/modeling/qs transfer function] between the sensed AHL concentration and the promoter activation deviates usually from the ideal one due to leakiness and crosstalk between different types of AHL molecules, regulatory proteins and promoters (we [https://2014.igem.org/Team:ETH_Zurich/expresults/qs#Quorum_Sensing characterized] it for [http://parts.igem.org/Part:BBa_R0062 Lux promoter (BBa_R0062)], [http://parts.igem.org/Part:BBa_R0079 Las promoter (BBa_R0079)] and [http://parts.igem.org/Part:BBa_R0071 Rhl promoter (BBa_R0071)]). To limit the risk of error propagation, we implemented a [http://parts.igem.org/Part:BBa_K1541000 riboregulated system].<br />
<br />
=== Computing ===<br />
<br />
[https://2014.igem.org/Team:ETH_Zurich/expresults#Integrases Integrases] compute an [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR logic gate] via single switching or double switching of a terminator. Their behavior depends on a set of [https://2014.igem.org/Team:ETH_Zurich/modeling/int parameters].<br />
<br />
=== Producing ===<br />
<br />
To propagate the computational results, the output has to be translated again into a quorum sensing molecule. Therefore a synthase is expressed to catalyze the production of a new AHL signal.<br />
<br />
=== Memorising ===<br />
<br />
Alongside the AHL synthase, GFP is produced according to the XOR truth table. The fluorescent protein acts both as long term memory for the colonies in ON-state and as visual signal, storing and showing the propagating pattern formation.<br />
<br />
=== Sending ===<br />
<br />
The produced AHL diffuses through channels on the memory unit to the rows below, becoming the input of the following information processing layer. The speed of this 'wiring' by directed diffusion determines the time needed for the pattern to emerge (as long as the channels are relatively long and diffusion is the time-limting step as compared to the actual computation itself). You can find out more information about diffusion and chip design on our [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel modeling page], our [https://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion_On_Chip experimental results page] and our [https://2014.igem.org/Team:ETH_Zurich/lab/chip chip page].<br />
<br />
<br />
<br />
[[File:ETH_Zurich_Information_Processing_chip.png|center|600px|thumb|'''Figure 2'''The information pathway in the project Mosai''coli'' on chip level. After initializing the signal, it propagates through the wells with directed diffusion. In each well, bacterial colonies have to be able to proceed in the information pathway: detecting (sensing), integrating (computing), producing and sending. These successive iterations leads to possible error propagation. Robustness is one major issue of our system.]]<br />
<br />
The overall process is summarized for the chip, or memory unit, level in figure 2.<br />
<br />
<html></article></html><br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/project/infoproTeam:ETH Zurich/project/infopro2014-10-18T02:48:33Z<p>Mbahls: /* Sending */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Information Processing}}<br />
<html><article></html><br />
<br />
== Information Processing ==<br />
<br />
<br />
=== About the track ===<br />
<br />
<br />
[https://2014.igem.org/Tracks/Information_Processing ''«Information Processing in iGEM covers a diverse range of projects. IP teams are not trying to solve a real world problem with practical applications, but to tackle an interesting problem that might otherwise not attract attention. Teams enter this track if they are attempting projects such as building elements of a biological computer, creating a game using biology or working on a signal processing challenges. Engineering ways to make biological systems perform computation is one of the core goals of synthetic biology. We generally work at the DNA level, engineering systems to function using BioBricks. In most biological systems, protein-protein interactions are where the majority of processing takes place. Being able to design proteins to accomplish computation would allow for systems to function on a much faster timescale than the current transcription-translation paradigm»'' (iGEM mainpage)]<br />
<br />
=== Why we chose this track ===<br />
<br />
With our project, Mosai''coli'', we investigate the emergence of complex patterns from simple mathematical rules. Such rules can be reduced to Boolean logic gates, in our case XOR gates. The computations were implemented with integrases, proteins modifing DNA between specific sites. These modifications in turn influence the expression of other proteins which can then indicate the previous change on the genetic level and chemically wire the information to the next cells, again on the protein or small-molecule level. This wiring of information and repeated information processing allows the construction of cellular automata and eventually biocomputers.<br />
<br />
=== The goal: Emergence of patterns via information processing===<br />
We implement a cellular automaton with bacterial cells containing our logic circuitry. Each bacterial colony serves as a core, computing an XOR gate. First, a sensor device detects the input, ''N''-acyl homoserine lactones (AHL). Then, the cell integrates this signal through a logic gate, performed by serine integrases by sensing on the protein level and acting on the DNA level. A necessary post-processing step allows then the production of a new AHL variant due to activated gene expression through the integrase. Meanwhile, green fluorescent protein (GFP) indicates the state of the colony and serves as a long-lasting visual read out. The produced AHL output-signal then propagates in a directed fashion through a millifluidic grid to the next bacterial colony. This iterative process faces the challenges of leakiness, cross-talk, protein-level computation and exact diffusion steps. This information pathway is shown in figure 1.<br />
<br />
<br />
[[File:ETH_Zurich2014_info_processing1.png|center|800px|thumb|'''Figure 1 The information pathway in the project Mosai''coli'' on a colony level.''' Each cell colony is part of a greater memory unit (left-hand side) and gets its input information from the neighboring colonies. The input is given in the form of two different AHLs, here named A and B. This information is then step-wise processed in each of the colonies: sensing - computing - producing - sending. The output molecules serve then as the input for the next colony (middle and left-hand side). This iterative process allows the information processing from the top row of the memory unit to the bottom by chemical wiring via diffusing signals and bio-computations inside of the colonies.]]<br />
<br />
<br />
<br />
=== The steps involved: From sensing to sending===<br />
<br />
=== Sensing ===<br />
<br />
Diffused AHL molecules are identified via the [https://2014.igem.org/Team:ETH_Zurich/expresults#Quorum_Sensing quorum sensing machinery]. The [https://2014.igem.org/Team:ETH_Zurich/modeling/qs transfer function] between the sensed AHL concentration and the promoter activation deviates usually from the ideal one due to leakiness and crosstalk between different types of AHL molecules, regulatory proteins and promoters (we [https://2014.igem.org/Team:ETH_Zurich/expresults/qs#Quorum_Sensing characterized] it for [http://parts.igem.org/Part:BBa_R0062 Lux promoter (BBa_R0062)], [http://parts.igem.org/Part:BBa_R0079 Las promoter (BBa_R0079)] and [http://parts.igem.org/Part:BBa_R0071 Rhl promoter (BBa_R0071)]). To limit the risk of error propagation, we implemented a [http://parts.igem.org/Part:BBa_K1541000 riboregulated system].<br />
<br />
=== Computing ===<br />
<br />
[https://2014.igem.org/Team:ETH_Zurich/expresults#Integrases Integrases] compute an [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR logic gate] via single switching or double switching of a terminator. Their behavior depends on a set of [https://2014.igem.org/Team:ETH_Zurich/modeling/int parameters].<br />
<br />
=== Producing ===<br />
<br />
To propagate the computational results, the output has to be translated again into a quorum sensing molecule. Therefore a synthase is expressed to catalyze the production of a new AHL signal.<br />
<br />
=== Memorising ===<br />
<br />
Alongside the AHL synthase, GFP is produced according to the XOR truth table. The fluorescent protein acts both as long term memory for the colonies in ON-state and as visual signal, storing and showing the propagating pattern formation.<br />
<br />
=== Sending ===<br />
<br />
The produced AHL diffuses through channels on the memory unit to the rows below, becoming the input of the following information processing layer. The speed of this 'wiring' by directed diffusion determines the time needed for the pattern to emerge (as long as the channels are relatively long and diffusion is the time-limting step as compared to the actual computation itself). You can find out more information about diffusion and chip design on our [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel modeling page], our [https://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion_On_Chip experimental results page] and our [https://2014.igem.org/Team:ETH_Zurich/lab/chip chip page].<br />
<br />
<br />
<br />
[[File:ETH_Zurich_Information_Processing_chip.png|center|600px|thumb|'''Figure 2'''The information pathway in the project Mosai''coli'' on chip level. After initializing the signal, it propagates through the wells with directed diffusion. In each well, bacterial colonies have to be able to proceed in the information pathway: detecting (sensing), integrating (computing), producing and sending. These successive iterations leads to possible error propagation. Robustness is one major issue of our system.]]<br />
<br />
<html></article></html><br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/project/infoproTeam:ETH Zurich/project/infopro2014-10-18T02:45:22Z<p>Mbahls: /* Memorising */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Information Processing}}<br />
<html><article></html><br />
<br />
== Information Processing ==<br />
<br />
<br />
=== About the track ===<br />
<br />
<br />
[https://2014.igem.org/Tracks/Information_Processing ''«Information Processing in iGEM covers a diverse range of projects. IP teams are not trying to solve a real world problem with practical applications, but to tackle an interesting problem that might otherwise not attract attention. Teams enter this track if they are attempting projects such as building elements of a biological computer, creating a game using biology or working on a signal processing challenges. Engineering ways to make biological systems perform computation is one of the core goals of synthetic biology. We generally work at the DNA level, engineering systems to function using BioBricks. In most biological systems, protein-protein interactions are where the majority of processing takes place. Being able to design proteins to accomplish computation would allow for systems to function on a much faster timescale than the current transcription-translation paradigm»'' (iGEM mainpage)]<br />
<br />
=== Why we chose this track ===<br />
<br />
With our project, Mosai''coli'', we investigate the emergence of complex patterns from simple mathematical rules. Such rules can be reduced to Boolean logic gates, in our case XOR gates. The computations were implemented with integrases, proteins modifing DNA between specific sites. These modifications in turn influence the expression of other proteins which can then indicate the previous change on the genetic level and chemically wire the information to the next cells, again on the protein or small-molecule level. This wiring of information and repeated information processing allows the construction of cellular automata and eventually biocomputers.<br />
<br />
=== The goal: Emergence of patterns via information processing===<br />
We implement a cellular automaton with bacterial cells containing our logic circuitry. Each bacterial colony serves as a core, computing an XOR gate. First, a sensor device detects the input, ''N''-acyl homoserine lactones (AHL). Then, the cell integrates this signal through a logic gate, performed by serine integrases by sensing on the protein level and acting on the DNA level. A necessary post-processing step allows then the production of a new AHL variant due to activated gene expression through the integrase. Meanwhile, green fluorescent protein (GFP) indicates the state of the colony and serves as a long-lasting visual read out. The produced AHL output-signal then propagates in a directed fashion through a millifluidic grid to the next bacterial colony. This iterative process faces the challenges of leakiness, cross-talk, protein-level computation and exact diffusion steps. This information pathway is shown in figure 1.<br />
<br />
<br />
[[File:ETH_Zurich2014_info_processing1.png|center|800px|thumb|'''Figure 1 The information pathway in the project Mosai''coli'' on a colony level.''' Each cell colony is part of a greater memory unit (left-hand side) and gets its input information from the neighboring colonies. The input is given in the form of two different AHLs, here named A and B. This information is then step-wise processed in each of the colonies: sensing - computing - producing - sending. The output molecules serve then as the input for the next colony (middle and left-hand side). This iterative process allows the information processing from the top row of the memory unit to the bottom by chemical wiring via diffusing signals and bio-computations inside of the colonies.]]<br />
<br />
<br />
<br />
=== The steps involved: From sensing to sending===<br />
<br />
=== Sensing ===<br />
<br />
Diffused AHL molecules are identified via the [https://2014.igem.org/Team:ETH_Zurich/expresults#Quorum_Sensing quorum sensing machinery]. The [https://2014.igem.org/Team:ETH_Zurich/modeling/qs transfer function] between the sensed AHL concentration and the promoter activation deviates usually from the ideal one due to leakiness and crosstalk between different types of AHL molecules, regulatory proteins and promoters (we [https://2014.igem.org/Team:ETH_Zurich/expresults/qs#Quorum_Sensing characterized] it for [http://parts.igem.org/Part:BBa_R0062 Lux promoter (BBa_R0062)], [http://parts.igem.org/Part:BBa_R0079 Las promoter (BBa_R0079)] and [http://parts.igem.org/Part:BBa_R0071 Rhl promoter (BBa_R0071)]). To limit the risk of error propagation, we implemented a [http://parts.igem.org/Part:BBa_K1541000 riboregulated system].<br />
<br />
=== Computing ===<br />
<br />
[https://2014.igem.org/Team:ETH_Zurich/expresults#Integrases Integrases] compute an [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR logic gate] via single switching or double switching of a terminator. Their behavior depends on a set of [https://2014.igem.org/Team:ETH_Zurich/modeling/int parameters].<br />
<br />
=== Producing ===<br />
<br />
To propagate the computational results, the output has to be translated again into a quorum sensing molecule. Therefore a synthase is expressed to catalyze the production of a new AHL signal.<br />
<br />
=== Memorising ===<br />
<br />
Alongside the AHL synthase, GFP is produced according to the XOR truth table. The fluorescent protein acts both as long term memory for the colonies in ON-state and as visual signal, storing and showing the propagating pattern formation.<br />
<br />
=== Sending ===<br />
<br />
The produced AHL diffuses through the channels to the rows below, becoming the input of the following layer. The speed of this directed diffusion determines the time needed from the pattern to emerge. You can find out more information about diffusion and chip design on our [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel modeling page], our [https://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion_On_Chip experimental results page] and our [https://2014.igem.org/Team:ETH_Zurich/lab/chip chip page].<br />
<br />
<br />
<br />
<br />
<br/><br />
<br/><br />
<br/><br />
[[File:ETH_Zurich_Information_Processing_chip.png|center|600px|thumb|'''Figure 2'''The information pathway in the project Mosai''coli'' on chip level. After initializing the signal, it propagates through the wells with directed diffusion. In each well, bacterial colonies have to be able to proceed in the information pathway: detecting (sensing), integrating (computing), producing and sending. These successive iterations leads to possible error propagation. Robustness is one major issue of our system.]]<br />
<br />
<html></article></html><br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/project/infoproTeam:ETH Zurich/project/infopro2014-10-18T02:43:19Z<p>Mbahls: /* Producing */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Information Processing}}<br />
<html><article></html><br />
<br />
== Information Processing ==<br />
<br />
<br />
=== About the track ===<br />
<br />
<br />
[https://2014.igem.org/Tracks/Information_Processing ''«Information Processing in iGEM covers a diverse range of projects. IP teams are not trying to solve a real world problem with practical applications, but to tackle an interesting problem that might otherwise not attract attention. Teams enter this track if they are attempting projects such as building elements of a biological computer, creating a game using biology or working on a signal processing challenges. Engineering ways to make biological systems perform computation is one of the core goals of synthetic biology. We generally work at the DNA level, engineering systems to function using BioBricks. In most biological systems, protein-protein interactions are where the majority of processing takes place. Being able to design proteins to accomplish computation would allow for systems to function on a much faster timescale than the current transcription-translation paradigm»'' (iGEM mainpage)]<br />
<br />
=== Why we chose this track ===<br />
<br />
With our project, Mosai''coli'', we investigate the emergence of complex patterns from simple mathematical rules. Such rules can be reduced to Boolean logic gates, in our case XOR gates. The computations were implemented with integrases, proteins modifing DNA between specific sites. These modifications in turn influence the expression of other proteins which can then indicate the previous change on the genetic level and chemically wire the information to the next cells, again on the protein or small-molecule level. This wiring of information and repeated information processing allows the construction of cellular automata and eventually biocomputers.<br />
<br />
=== The goal: Emergence of patterns via information processing===<br />
We implement a cellular automaton with bacterial cells containing our logic circuitry. Each bacterial colony serves as a core, computing an XOR gate. First, a sensor device detects the input, ''N''-acyl homoserine lactones (AHL). Then, the cell integrates this signal through a logic gate, performed by serine integrases by sensing on the protein level and acting on the DNA level. A necessary post-processing step allows then the production of a new AHL variant due to activated gene expression through the integrase. Meanwhile, green fluorescent protein (GFP) indicates the state of the colony and serves as a long-lasting visual read out. The produced AHL output-signal then propagates in a directed fashion through a millifluidic grid to the next bacterial colony. This iterative process faces the challenges of leakiness, cross-talk, protein-level computation and exact diffusion steps. This information pathway is shown in figure 1.<br />
<br />
<br />
[[File:ETH_Zurich2014_info_processing1.png|center|800px|thumb|'''Figure 1 The information pathway in the project Mosai''coli'' on a colony level.''' Each cell colony is part of a greater memory unit (left-hand side) and gets its input information from the neighboring colonies. The input is given in the form of two different AHLs, here named A and B. This information is then step-wise processed in each of the colonies: sensing - computing - producing - sending. The output molecules serve then as the input for the next colony (middle and left-hand side). This iterative process allows the information processing from the top row of the memory unit to the bottom by chemical wiring via diffusing signals and bio-computations inside of the colonies.]]<br />
<br />
<br />
<br />
=== The steps involved: From sensing to sending===<br />
<br />
=== Sensing ===<br />
<br />
Diffused AHL molecules are identified via the [https://2014.igem.org/Team:ETH_Zurich/expresults#Quorum_Sensing quorum sensing machinery]. The [https://2014.igem.org/Team:ETH_Zurich/modeling/qs transfer function] between the sensed AHL concentration and the promoter activation deviates usually from the ideal one due to leakiness and crosstalk between different types of AHL molecules, regulatory proteins and promoters (we [https://2014.igem.org/Team:ETH_Zurich/expresults/qs#Quorum_Sensing characterized] it for [http://parts.igem.org/Part:BBa_R0062 Lux promoter (BBa_R0062)], [http://parts.igem.org/Part:BBa_R0079 Las promoter (BBa_R0079)] and [http://parts.igem.org/Part:BBa_R0071 Rhl promoter (BBa_R0071)]). To limit the risk of error propagation, we implemented a [http://parts.igem.org/Part:BBa_K1541000 riboregulated system].<br />
<br />
=== Computing ===<br />
<br />
[https://2014.igem.org/Team:ETH_Zurich/expresults#Integrases Integrases] compute an [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR logic gate] via single switching or double switching of a terminator. Their behavior depends on a set of [https://2014.igem.org/Team:ETH_Zurich/modeling/int parameters].<br />
<br />
=== Producing ===<br />
<br />
To propagate the computational results, the output has to be translated again into a quorum sensing molecule. Therefore a synthase is expressed to catalyze the production of a new AHL signal.<br />
<br />
=== Memorising ===<br />
<br />
Alongside the AHL synthase, GFP is produced according to the XOR output. Th fluorescent protein acts both as long term memory for the colony state and as visual signal, storing and showing the progressive pattern formation.<br />
<br />
=== Sending ===<br />
<br />
The produced AHL diffuses through the channels to the rows below, becoming the input of the following layer. The speed of this directed diffusion determines the time needed from the pattern to emerge. You can find out more information about diffusion and chip design on our [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel modeling page], our [https://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion_On_Chip experimental results page] and our [https://2014.igem.org/Team:ETH_Zurich/lab/chip chip page].<br />
<br />
<br />
<br />
<br />
<br/><br />
<br/><br />
<br/><br />
[[File:ETH_Zurich_Information_Processing_chip.png|center|600px|thumb|'''Figure 2'''The information pathway in the project Mosai''coli'' on chip level. After initializing the signal, it propagates through the wells with directed diffusion. In each well, bacterial colonies have to be able to proceed in the information pathway: detecting (sensing), integrating (computing), producing and sending. These successive iterations leads to possible error propagation. Robustness is one major issue of our system.]]<br />
<br />
<html></article></html><br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/project/infoproTeam:ETH Zurich/project/infopro2014-10-18T02:42:51Z<p>Mbahls: /* Producing */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Information Processing}}<br />
<html><article></html><br />
<br />
== Information Processing ==<br />
<br />
<br />
=== About the track ===<br />
<br />
<br />
[https://2014.igem.org/Tracks/Information_Processing ''«Information Processing in iGEM covers a diverse range of projects. IP teams are not trying to solve a real world problem with practical applications, but to tackle an interesting problem that might otherwise not attract attention. Teams enter this track if they are attempting projects such as building elements of a biological computer, creating a game using biology or working on a signal processing challenges. Engineering ways to make biological systems perform computation is one of the core goals of synthetic biology. We generally work at the DNA level, engineering systems to function using BioBricks. In most biological systems, protein-protein interactions are where the majority of processing takes place. Being able to design proteins to accomplish computation would allow for systems to function on a much faster timescale than the current transcription-translation paradigm»'' (iGEM mainpage)]<br />
<br />
=== Why we chose this track ===<br />
<br />
With our project, Mosai''coli'', we investigate the emergence of complex patterns from simple mathematical rules. Such rules can be reduced to Boolean logic gates, in our case XOR gates. The computations were implemented with integrases, proteins modifing DNA between specific sites. These modifications in turn influence the expression of other proteins which can then indicate the previous change on the genetic level and chemically wire the information to the next cells, again on the protein or small-molecule level. This wiring of information and repeated information processing allows the construction of cellular automata and eventually biocomputers.<br />
<br />
=== The goal: Emergence of patterns via information processing===<br />
We implement a cellular automaton with bacterial cells containing our logic circuitry. Each bacterial colony serves as a core, computing an XOR gate. First, a sensor device detects the input, ''N''-acyl homoserine lactones (AHL). Then, the cell integrates this signal through a logic gate, performed by serine integrases by sensing on the protein level and acting on the DNA level. A necessary post-processing step allows then the production of a new AHL variant due to activated gene expression through the integrase. Meanwhile, green fluorescent protein (GFP) indicates the state of the colony and serves as a long-lasting visual read out. The produced AHL output-signal then propagates in a directed fashion through a millifluidic grid to the next bacterial colony. This iterative process faces the challenges of leakiness, cross-talk, protein-level computation and exact diffusion steps. This information pathway is shown in figure 1.<br />
<br />
<br />
[[File:ETH_Zurich2014_info_processing1.png|center|800px|thumb|'''Figure 1 The information pathway in the project Mosai''coli'' on a colony level.''' Each cell colony is part of a greater memory unit (left-hand side) and gets its input information from the neighboring colonies. The input is given in the form of two different AHLs, here named A and B. This information is then step-wise processed in each of the colonies: sensing - computing - producing - sending. The output molecules serve then as the input for the next colony (middle and left-hand side). This iterative process allows the information processing from the top row of the memory unit to the bottom by chemical wiring via diffusing signals and bio-computations inside of the colonies.]]<br />
<br />
<br />
<br />
=== The steps involved: From sensing to sending===<br />
<br />
=== Sensing ===<br />
<br />
Diffused AHL molecules are identified via the [https://2014.igem.org/Team:ETH_Zurich/expresults#Quorum_Sensing quorum sensing machinery]. The [https://2014.igem.org/Team:ETH_Zurich/modeling/qs transfer function] between the sensed AHL concentration and the promoter activation deviates usually from the ideal one due to leakiness and crosstalk between different types of AHL molecules, regulatory proteins and promoters (we [https://2014.igem.org/Team:ETH_Zurich/expresults/qs#Quorum_Sensing characterized] it for [http://parts.igem.org/Part:BBa_R0062 Lux promoter (BBa_R0062)], [http://parts.igem.org/Part:BBa_R0079 Las promoter (BBa_R0079)] and [http://parts.igem.org/Part:BBa_R0071 Rhl promoter (BBa_R0071)]). To limit the risk of error propagation, we implemented a [http://parts.igem.org/Part:BBa_K1541000 riboregulated system].<br />
<br />
=== Computing ===<br />
<br />
[https://2014.igem.org/Team:ETH_Zurich/expresults#Integrases Integrases] compute an [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR logic gate] via single switching or double switching of a terminator. Their behavior depends on a set of [https://2014.igem.org/Team:ETH_Zurich/modeling/int parameters].<br />
<br />
=== Producing ===<br />
<br />
To propagate the computation results, the output has to be translated again into a quorum sensing molecule. Therefore a synthase is expressed to catalyze the production of a new AHL signal.<br />
<br />
=== Memorising ===<br />
<br />
Alongside the AHL synthase, GFP is produced according to the XOR output. Th fluorescent protein acts both as long term memory for the colony state and as visual signal, storing and showing the progressive pattern formation.<br />
<br />
=== Sending ===<br />
<br />
The produced AHL diffuses through the channels to the rows below, becoming the input of the following layer. The speed of this directed diffusion determines the time needed from the pattern to emerge. You can find out more information about diffusion and chip design on our [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel modeling page], our [https://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion_On_Chip experimental results page] and our [https://2014.igem.org/Team:ETH_Zurich/lab/chip chip page].<br />
<br />
<br />
<br />
<br />
<br/><br />
<br/><br />
<br/><br />
[[File:ETH_Zurich_Information_Processing_chip.png|center|600px|thumb|'''Figure 2'''The information pathway in the project Mosai''coli'' on chip level. After initializing the signal, it propagates through the wells with directed diffusion. In each well, bacterial colonies have to be able to proceed in the information pathway: detecting (sensing), integrating (computing), producing and sending. These successive iterations leads to possible error propagation. Robustness is one major issue of our system.]]<br />
<br />
<html></article></html><br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/project/infoproTeam:ETH Zurich/project/infopro2014-10-18T02:41:14Z<p>Mbahls: /* The steps involved: From sensing to sending */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Information Processing}}<br />
<html><article></html><br />
<br />
== Information Processing ==<br />
<br />
<br />
=== About the track ===<br />
<br />
<br />
[https://2014.igem.org/Tracks/Information_Processing ''«Information Processing in iGEM covers a diverse range of projects. IP teams are not trying to solve a real world problem with practical applications, but to tackle an interesting problem that might otherwise not attract attention. Teams enter this track if they are attempting projects such as building elements of a biological computer, creating a game using biology or working on a signal processing challenges. Engineering ways to make biological systems perform computation is one of the core goals of synthetic biology. We generally work at the DNA level, engineering systems to function using BioBricks. In most biological systems, protein-protein interactions are where the majority of processing takes place. Being able to design proteins to accomplish computation would allow for systems to function on a much faster timescale than the current transcription-translation paradigm»'' (iGEM mainpage)]<br />
<br />
=== Why we chose this track ===<br />
<br />
With our project, Mosai''coli'', we investigate the emergence of complex patterns from simple mathematical rules. Such rules can be reduced to Boolean logic gates, in our case XOR gates. The computations were implemented with integrases, proteins modifing DNA between specific sites. These modifications in turn influence the expression of other proteins which can then indicate the previous change on the genetic level and chemically wire the information to the next cells, again on the protein or small-molecule level. This wiring of information and repeated information processing allows the construction of cellular automata and eventually biocomputers.<br />
<br />
=== The goal: Emergence of patterns via information processing===<br />
We implement a cellular automaton with bacterial cells containing our logic circuitry. Each bacterial colony serves as a core, computing an XOR gate. First, a sensor device detects the input, ''N''-acyl homoserine lactones (AHL). Then, the cell integrates this signal through a logic gate, performed by serine integrases by sensing on the protein level and acting on the DNA level. A necessary post-processing step allows then the production of a new AHL variant due to activated gene expression through the integrase. Meanwhile, green fluorescent protein (GFP) indicates the state of the colony and serves as a long-lasting visual read out. The produced AHL output-signal then propagates in a directed fashion through a millifluidic grid to the next bacterial colony. This iterative process faces the challenges of leakiness, cross-talk, protein-level computation and exact diffusion steps. This information pathway is shown in figure 1.<br />
<br />
<br />
[[File:ETH_Zurich2014_info_processing1.png|center|800px|thumb|'''Figure 1 The information pathway in the project Mosai''coli'' on a colony level.''' Each cell colony is part of a greater memory unit (left-hand side) and gets its input information from the neighboring colonies. The input is given in the form of two different AHLs, here named A and B. This information is then step-wise processed in each of the colonies: sensing - computing - producing - sending. The output molecules serve then as the input for the next colony (middle and left-hand side). This iterative process allows the information processing from the top row of the memory unit to the bottom by chemical wiring via diffusing signals and bio-computations inside of the colonies.]]<br />
<br />
<br />
<br />
=== The steps involved: From sensing to sending===<br />
<br />
=== Sensing ===<br />
<br />
Diffused AHL molecules are identified via the [https://2014.igem.org/Team:ETH_Zurich/expresults#Quorum_Sensing quorum sensing machinery]. The [https://2014.igem.org/Team:ETH_Zurich/modeling/qs transfer function] between the sensed AHL concentration and the promoter activation deviates usually from the ideal one due to leakiness and crosstalk between different types of AHL molecules, regulatory proteins and promoters (we [https://2014.igem.org/Team:ETH_Zurich/expresults/qs#Quorum_Sensing characterized] it for [http://parts.igem.org/Part:BBa_R0062 Lux promoter (BBa_R0062)], [http://parts.igem.org/Part:BBa_R0079 Las promoter (BBa_R0079)] and [http://parts.igem.org/Part:BBa_R0071 Rhl promoter (BBa_R0071)]). To limit the risk of error propagation, we implemented a [http://parts.igem.org/Part:BBa_K1541000 riboregulated system].<br />
<br />
=== Computing ===<br />
<br />
[https://2014.igem.org/Team:ETH_Zurich/expresults#Integrases Integrases] compute an [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR logic gate] via single switching or double switching of a terminator. Their behavior depends on a set of [https://2014.igem.org/Team:ETH_Zurich/modeling/int parameters].<br />
<br />
=== Producing ===<br />
<br />
To propagate the computation results, the output has to be translated again into a quorum sensing molecule. Therefore a synthase is expressed to produce a new AHL signal.<br />
<br />
=== Memorising ===<br />
<br />
Alongside the AHL synthase, GFP is produced according to the XOR output. Th fluorescent protein acts both as long term memory for the colony state and as visual signal, storing and showing the progressive pattern formation.<br />
<br />
=== Sending ===<br />
<br />
The produced AHL diffuses through the channels to the rows below, becoming the input of the following layer. The speed of this directed diffusion determines the time needed from the pattern to emerge. You can find out more information about diffusion and chip design on our [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel modeling page], our [https://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion_On_Chip experimental results page] and our [https://2014.igem.org/Team:ETH_Zurich/lab/chip chip page].<br />
<br />
<br />
<br />
<br />
<br/><br />
<br/><br />
<br/><br />
[[File:ETH_Zurich_Information_Processing_chip.png|center|600px|thumb|'''Figure 2'''The information pathway in the project Mosai''coli'' on chip level. After initializing the signal, it propagates through the wells with directed diffusion. In each well, bacterial colonies have to be able to proceed in the information pathway: detecting (sensing), integrating (computing), producing and sending. These successive iterations leads to possible error propagation. Robustness is one major issue of our system.]]<br />
<br />
<html></article></html><br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/project/infoproTeam:ETH Zurich/project/infopro2014-10-18T02:40:58Z<p>Mbahls: /* Sensing */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Information Processing}}<br />
<html><article></html><br />
<br />
== Information Processing ==<br />
<br />
<br />
=== About the track ===<br />
<br />
<br />
[https://2014.igem.org/Tracks/Information_Processing ''«Information Processing in iGEM covers a diverse range of projects. IP teams are not trying to solve a real world problem with practical applications, but to tackle an interesting problem that might otherwise not attract attention. Teams enter this track if they are attempting projects such as building elements of a biological computer, creating a game using biology or working on a signal processing challenges. Engineering ways to make biological systems perform computation is one of the core goals of synthetic biology. We generally work at the DNA level, engineering systems to function using BioBricks. In most biological systems, protein-protein interactions are where the majority of processing takes place. Being able to design proteins to accomplish computation would allow for systems to function on a much faster timescale than the current transcription-translation paradigm»'' (iGEM mainpage)]<br />
<br />
=== Why we chose this track ===<br />
<br />
With our project, Mosai''coli'', we investigate the emergence of complex patterns from simple mathematical rules. Such rules can be reduced to Boolean logic gates, in our case XOR gates. The computations were implemented with integrases, proteins modifing DNA between specific sites. These modifications in turn influence the expression of other proteins which can then indicate the previous change on the genetic level and chemically wire the information to the next cells, again on the protein or small-molecule level. This wiring of information and repeated information processing allows the construction of cellular automata and eventually biocomputers.<br />
<br />
=== The goal: Emergence of patterns via information processing===<br />
We implement a cellular automaton with bacterial cells containing our logic circuitry. Each bacterial colony serves as a core, computing an XOR gate. First, a sensor device detects the input, ''N''-acyl homoserine lactones (AHL). Then, the cell integrates this signal through a logic gate, performed by serine integrases by sensing on the protein level and acting on the DNA level. A necessary post-processing step allows then the production of a new AHL variant due to activated gene expression through the integrase. Meanwhile, green fluorescent protein (GFP) indicates the state of the colony and serves as a long-lasting visual read out. The produced AHL output-signal then propagates in a directed fashion through a millifluidic grid to the next bacterial colony. This iterative process faces the challenges of leakiness, cross-talk, protein-level computation and exact diffusion steps. This information pathway is shown in figure 1.<br />
<br />
<br />
[[File:ETH_Zurich2014_info_processing1.png|center|800px|thumb|'''Figure 1 The information pathway in the project Mosai''coli'' on a colony level.''' Each cell colony is part of a greater memory unit (left-hand side) and gets its input information from the neighboring colonies. The input is given in the form of two different AHLs, here named A and B. This information is then step-wise processed in each of the colonies: sensing - computing - producing - sending. The output molecules serve then as the input for the next colony (middle and left-hand side). This iterative process allows the information processing from the top row of the memory unit to the bottom by chemical wiring via diffusing signals and bio-computations inside of the colonies.]]<br />
<br />
=== The steps involved: From sensing to sending===<br />
<br />
=== Sensing ===<br />
<br />
Diffused AHL molecules are identified via the [https://2014.igem.org/Team:ETH_Zurich/expresults#Quorum_Sensing quorum sensing machinery]. The [https://2014.igem.org/Team:ETH_Zurich/modeling/qs transfer function] between the sensed AHL concentration and the promoter activation deviates usually from the ideal one due to leakiness and crosstalk between different types of AHL molecules, regulatory proteins and promoters (we [https://2014.igem.org/Team:ETH_Zurich/expresults/qs#Quorum_Sensing characterized] it for [http://parts.igem.org/Part:BBa_R0062 Lux promoter (BBa_R0062)], [http://parts.igem.org/Part:BBa_R0079 Las promoter (BBa_R0079)] and [http://parts.igem.org/Part:BBa_R0071 Rhl promoter (BBa_R0071)]). To limit the risk of error propagation, we implemented a [http://parts.igem.org/Part:BBa_K1541000 riboregulated system].<br />
<br />
=== Computing ===<br />
<br />
[https://2014.igem.org/Team:ETH_Zurich/expresults#Integrases Integrases] compute an [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR logic gate] via single switching or double switching of a terminator. Their behavior depends on a set of [https://2014.igem.org/Team:ETH_Zurich/modeling/int parameters].<br />
<br />
=== Producing ===<br />
<br />
To propagate the computation results, the output has to be translated again into a quorum sensing molecule. Therefore a synthase is expressed to produce a new AHL signal.<br />
<br />
=== Memorising ===<br />
<br />
Alongside the AHL synthase, GFP is produced according to the XOR output. Th fluorescent protein acts both as long term memory for the colony state and as visual signal, storing and showing the progressive pattern formation.<br />
<br />
=== Sending ===<br />
<br />
The produced AHL diffuses through the channels to the rows below, becoming the input of the following layer. The speed of this directed diffusion determines the time needed from the pattern to emerge. You can find out more information about diffusion and chip design on our [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel modeling page], our [https://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion_On_Chip experimental results page] and our [https://2014.igem.org/Team:ETH_Zurich/lab/chip chip page].<br />
<br />
<br />
<br />
<br />
<br/><br />
<br/><br />
<br/><br />
[[File:ETH_Zurich_Information_Processing_chip.png|center|600px|thumb|'''Figure 2'''The information pathway in the project Mosai''coli'' on chip level. After initializing the signal, it propagates through the wells with directed diffusion. In each well, bacterial colonies have to be able to proceed in the information pathway: detecting (sensing), integrating (computing), producing and sending. These successive iterations leads to possible error propagation. Robustness is one major issue of our system.]]<br />
<br />
<html></article></html><br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/project/infoproTeam:ETH Zurich/project/infopro2014-10-18T02:40:01Z<p>Mbahls: /* Sensing */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Information Processing}}<br />
<html><article></html><br />
<br />
== Information Processing ==<br />
<br />
<br />
=== About the track ===<br />
<br />
<br />
[https://2014.igem.org/Tracks/Information_Processing ''«Information Processing in iGEM covers a diverse range of projects. IP teams are not trying to solve a real world problem with practical applications, but to tackle an interesting problem that might otherwise not attract attention. Teams enter this track if they are attempting projects such as building elements of a biological computer, creating a game using biology or working on a signal processing challenges. Engineering ways to make biological systems perform computation is one of the core goals of synthetic biology. We generally work at the DNA level, engineering systems to function using BioBricks. In most biological systems, protein-protein interactions are where the majority of processing takes place. Being able to design proteins to accomplish computation would allow for systems to function on a much faster timescale than the current transcription-translation paradigm»'' (iGEM mainpage)]<br />
<br />
=== Why we chose this track ===<br />
<br />
With our project, Mosai''coli'', we investigate the emergence of complex patterns from simple mathematical rules. Such rules can be reduced to Boolean logic gates, in our case XOR gates. The computations were implemented with integrases, proteins modifing DNA between specific sites. These modifications in turn influence the expression of other proteins which can then indicate the previous change on the genetic level and chemically wire the information to the next cells, again on the protein or small-molecule level. This wiring of information and repeated information processing allows the construction of cellular automata and eventually biocomputers.<br />
<br />
=== The goal: Emergence of patterns via information processing===<br />
We implement a cellular automaton with bacterial cells containing our logic circuitry. Each bacterial colony serves as a core, computing an XOR gate. First, a sensor device detects the input, ''N''-acyl homoserine lactones (AHL). Then, the cell integrates this signal through a logic gate, performed by serine integrases by sensing on the protein level and acting on the DNA level. A necessary post-processing step allows then the production of a new AHL variant due to activated gene expression through the integrase. Meanwhile, green fluorescent protein (GFP) indicates the state of the colony and serves as a long-lasting visual read out. The produced AHL output-signal then propagates in a directed fashion through a millifluidic grid to the next bacterial colony. This iterative process faces the challenges of leakiness, cross-talk, protein-level computation and exact diffusion steps. This information pathway is shown in figure 1.<br />
<br />
<br />
[[File:ETH_Zurich2014_info_processing1.png|center|800px|thumb|'''Figure 1 The information pathway in the project Mosai''coli'' on a colony level.''' Each cell colony is part of a greater memory unit (left-hand side) and gets its input information from the neighboring colonies. The input is given in the form of two different AHLs, here named A and B. This information is then step-wise processed in each of the colonies: sensing - computing - producing - sending. The output molecules serve then as the input for the next colony (middle and left-hand side). This iterative process allows the information processing from the top row of the memory unit to the bottom by chemical wiring via diffusing signals and bio-computations inside of the colonies.]]<br />
<br />
=== The steps involved: From sensing to sending===<br />
<br />
=== Sensing ===<br />
<br />
Diffused AHL molecules are identified via the [https://2014.igem.org/Team:ETH_Zurich/expresults#Quorum_Sensing quorum sensing machinery]. The [https://2014.igem.org/Team:ETH_Zurich/modeling/qs transfer function] between the sensed AHL concentration and the promoter activation deviates usually from the ideal one due to leakiness and crosstalk between different types of AHL molecules, regulatory proteins and promoters (we characterized it for [http://parts.igem.org/Part:BBa_R0062 Lux promoter (BBa_R0062)], [http://parts.igem.org/Part:BBa_R0079 Las promoter (BBa_R0079)] and [http://parts.igem.org/Part:BBa_R0071 Rhl promoter (BBa_R0071)]). To limit the risk of error propagation, we implemented a [http://parts.igem.org/Part:BBa_K1541000 riboregulated system].<br />
<br />
=== Computing ===<br />
<br />
[https://2014.igem.org/Team:ETH_Zurich/expresults#Integrases Integrases] compute an [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR logic gate] via single switching or double switching of a terminator. Their behavior depends on a set of [https://2014.igem.org/Team:ETH_Zurich/modeling/int parameters].<br />
<br />
=== Producing ===<br />
<br />
To propagate the computation results, the output has to be translated again into a quorum sensing molecule. Therefore a synthase is expressed to produce a new AHL signal.<br />
<br />
=== Memorising ===<br />
<br />
Alongside the AHL synthase, GFP is produced according to the XOR output. Th fluorescent protein acts both as long term memory for the colony state and as visual signal, storing and showing the progressive pattern formation.<br />
<br />
=== Sending ===<br />
<br />
The produced AHL diffuses through the channels to the rows below, becoming the input of the following layer. The speed of this directed diffusion determines the time needed from the pattern to emerge. You can find out more information about diffusion and chip design on our [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel modeling page], our [https://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion_On_Chip experimental results page] and our [https://2014.igem.org/Team:ETH_Zurich/lab/chip chip page].<br />
<br />
<br />
<br />
<br />
<br/><br />
<br/><br />
<br/><br />
[[File:ETH_Zurich_Information_Processing_chip.png|center|600px|thumb|'''Figure 2'''The information pathway in the project Mosai''coli'' on chip level. After initializing the signal, it propagates through the wells with directed diffusion. In each well, bacterial colonies have to be able to proceed in the information pathway: detecting (sensing), integrating (computing), producing and sending. These successive iterations leads to possible error propagation. Robustness is one major issue of our system.]]<br />
<br />
<html></article></html><br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/project/infoproTeam:ETH Zurich/project/infopro2014-10-18T02:38:21Z<p>Mbahls: /* Sensing */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Information Processing}}<br />
<html><article></html><br />
<br />
== Information Processing ==<br />
<br />
<br />
=== About the track ===<br />
<br />
<br />
[https://2014.igem.org/Tracks/Information_Processing ''«Information Processing in iGEM covers a diverse range of projects. IP teams are not trying to solve a real world problem with practical applications, but to tackle an interesting problem that might otherwise not attract attention. Teams enter this track if they are attempting projects such as building elements of a biological computer, creating a game using biology or working on a signal processing challenges. Engineering ways to make biological systems perform computation is one of the core goals of synthetic biology. We generally work at the DNA level, engineering systems to function using BioBricks. In most biological systems, protein-protein interactions are where the majority of processing takes place. Being able to design proteins to accomplish computation would allow for systems to function on a much faster timescale than the current transcription-translation paradigm»'' (iGEM mainpage)]<br />
<br />
=== Why we chose this track ===<br />
<br />
With our project, Mosai''coli'', we investigate the emergence of complex patterns from simple mathematical rules. Such rules can be reduced to Boolean logic gates, in our case XOR gates. The computations were implemented with integrases, proteins modifing DNA between specific sites. These modifications in turn influence the expression of other proteins which can then indicate the previous change on the genetic level and chemically wire the information to the next cells, again on the protein or small-molecule level. This wiring of information and repeated information processing allows the construction of cellular automata and eventually biocomputers.<br />
<br />
=== The goal: Emergence of patterns via information processing===<br />
We implement a cellular automaton with bacterial cells containing our logic circuitry. Each bacterial colony serves as a core, computing an XOR gate. First, a sensor device detects the input, ''N''-acyl homoserine lactones (AHL). Then, the cell integrates this signal through a logic gate, performed by serine integrases by sensing on the protein level and acting on the DNA level. A necessary post-processing step allows then the production of a new AHL variant due to activated gene expression through the integrase. Meanwhile, green fluorescent protein (GFP) indicates the state of the colony and serves as a long-lasting visual read out. The produced AHL output-signal then propagates in a directed fashion through a millifluidic grid to the next bacterial colony. This iterative process faces the challenges of leakiness, cross-talk, protein-level computation and exact diffusion steps. This information pathway is shown in figure 1.<br />
<br />
<br />
[[File:ETH_Zurich2014_info_processing1.png|center|800px|thumb|'''Figure 1 The information pathway in the project Mosai''coli'' on a colony level.''' Each cell colony is part of a greater memory unit (left-hand side) and gets its input information from the neighboring colonies. The input is given in the form of two different AHLs, here named A and B. This information is then step-wise processed in each of the colonies: sensing - computing - producing - sending. The output molecules serve then as the input for the next colony (middle and left-hand side). This iterative process allows the information processing from the top row of the memory unit to the bottom by chemical wiring via diffusing signals and bio-computations inside of the colonies.]]<br />
<br />
=== The steps involved: From sensing to sending===<br />
<br />
=== Sensing ===<br />
<br />
Diffused AHL molecules are identified via the [https://2014.igem.org/Team:ETH_Zurich/expresults#Quorum_Sensing quorum sensing machinery]. The [https://2014.igem.org/Team:ETH_Zurich/modeling/qs transfer function] between concentration of sensed AHL to promoter activation deviates from the ideal one due to leakiness and crosstalk between HSL molecules, regulatory proteins and promoters (we characterized it for [http://parts.igem.org/Part:BBa_R0062 Lux promoter (BBa_R0062)], [http://parts.igem.org/Part:BBa_R0079 Las promoter (BBa_R0079)] and [http://parts.igem.org/Part:BBa_R0071 Rhl promoter (BBa_R0071)]). To limit the risk of error propagation, we implemented a [http://parts.igem.org/Part:BBa_K1541000 riboregulated system].<br />
<br />
=== Computing ===<br />
<br />
[https://2014.igem.org/Team:ETH_Zurich/expresults#Integrases Integrases] compute an [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR logic gate] via single switching or double switching of a terminator. Their behavior depends on a set of [https://2014.igem.org/Team:ETH_Zurich/modeling/int parameters].<br />
<br />
=== Producing ===<br />
<br />
To propagate the computation results, the output has to be translated again into a quorum sensing molecule. Therefore a synthase is expressed to produce a new AHL signal.<br />
<br />
=== Memorising ===<br />
<br />
Alongside the AHL synthase, GFP is produced according to the XOR output. Th fluorescent protein acts both as long term memory for the colony state and as visual signal, storing and showing the progressive pattern formation.<br />
<br />
=== Sending ===<br />
<br />
The produced AHL diffuses through the channels to the rows below, becoming the input of the following layer. The speed of this directed diffusion determines the time needed from the pattern to emerge. You can find out more information about diffusion and chip design on our [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel modeling page], our [https://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion_On_Chip experimental results page] and our [https://2014.igem.org/Team:ETH_Zurich/lab/chip chip page].<br />
<br />
<br />
<br />
<br />
<br/><br />
<br/><br />
<br/><br />
[[File:ETH_Zurich_Information_Processing_chip.png|center|600px|thumb|'''Figure 2'''The information pathway in the project Mosai''coli'' on chip level. After initializing the signal, it propagates through the wells with directed diffusion. In each well, bacterial colonies have to be able to proceed in the information pathway: detecting (sensing), integrating (computing), producing and sending. These successive iterations leads to possible error propagation. Robustness is one major issue of our system.]]<br />
<br />
<html></article></html><br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/project/infoproTeam:ETH Zurich/project/infopro2014-10-18T02:37:16Z<p>Mbahls: /* The goal: Emergence of patterns via information processing */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Information Processing}}<br />
<html><article></html><br />
<br />
== Information Processing ==<br />
<br />
<br />
=== About the track ===<br />
<br />
<br />
[https://2014.igem.org/Tracks/Information_Processing ''«Information Processing in iGEM covers a diverse range of projects. IP teams are not trying to solve a real world problem with practical applications, but to tackle an interesting problem that might otherwise not attract attention. Teams enter this track if they are attempting projects such as building elements of a biological computer, creating a game using biology or working on a signal processing challenges. Engineering ways to make biological systems perform computation is one of the core goals of synthetic biology. We generally work at the DNA level, engineering systems to function using BioBricks. In most biological systems, protein-protein interactions are where the majority of processing takes place. Being able to design proteins to accomplish computation would allow for systems to function on a much faster timescale than the current transcription-translation paradigm»'' (iGEM mainpage)]<br />
<br />
=== Why we chose this track ===<br />
<br />
With our project, Mosai''coli'', we investigate the emergence of complex patterns from simple mathematical rules. Such rules can be reduced to Boolean logic gates, in our case XOR gates. The computations were implemented with integrases, proteins modifing DNA between specific sites. These modifications in turn influence the expression of other proteins which can then indicate the previous change on the genetic level and chemically wire the information to the next cells, again on the protein or small-molecule level. This wiring of information and repeated information processing allows the construction of cellular automata and eventually biocomputers.<br />
<br />
=== The goal: Emergence of patterns via information processing===<br />
We implement a cellular automaton with bacterial cells containing our logic circuitry. Each bacterial colony serves as a core, computing an XOR gate. First, a sensor device detects the input, ''N''-acyl homoserine lactones (AHL). Then, the cell integrates this signal through a logic gate, performed by serine integrases by sensing on the protein level and acting on the DNA level. A necessary post-processing step allows then the production of a new AHL variant due to activated gene expression through the integrase. Meanwhile, green fluorescent protein (GFP) indicates the state of the colony and serves as a long-lasting visual read out. The produced AHL output-signal then propagates in a directed fashion through a millifluidic grid to the next bacterial colony. This iterative process faces the challenges of leakiness, cross-talk, protein-level computation and exact diffusion steps. This information pathway is shown in figure 1.<br />
<br />
<br />
[[File:ETH_Zurich2014_info_processing1.png|center|800px|thumb|'''Figure 1 The information pathway in the project Mosai''coli'' on a colony level.''' Each cell colony is part of a greater memory unit (left-hand side) and gets its input information from the neighboring colonies. The input is given in the form of two different AHLs, here named A and B. This information is then step-wise processed in each of the colonies: sensing - computing - producing - sending. The output molecules serve then as the input for the next colony (middle and left-hand side). This iterative process allows the information processing from the top row of the memory unit to the bottom by chemical wiring via diffusing signals and bio-computations inside of the colonies.]]<br />
<br />
=== The steps involved: From sensing to sending===<br />
<br />
=== Sensing ===<br />
<br />
Diffused HSL molecules are identified via [https://2014.igem.org/Team:ETH_Zurich/expresults#Quorum_Sensing quorum sensing machinery]. The [https://2014.igem.org/Team:ETH_Zurich/modeling/qs transfer function] between concentration of sensed HSL to promoter activation deviates from the ideal one due to leakiness and crosstalk between HSL molecules, regulatory proteins and promoters (we characterized it for [http://parts.igem.org/Part:BBa_R0062 Lux promoter (BBa_R0062)], [http://parts.igem.org/Part:BBa_R0079 Las promoter (BBa_R0079)] and [http://parts.igem.org/Part:BBa_R0071 Rhl promoter (BBa_R0071)]). To limit the risk of error propagation, we implemented a [http://parts.igem.org/Part:BBa_K1541000 riboregulated system].<br />
<br />
=== Computing ===<br />
<br />
[https://2014.igem.org/Team:ETH_Zurich/expresults#Integrases Integrases] compute an [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR logic gate] via single switching or double switching of a terminator. Their behavior depends on a set of [https://2014.igem.org/Team:ETH_Zurich/modeling/int parameters].<br />
<br />
=== Producing ===<br />
<br />
To propagate the computation results, the output has to be translated again into a quorum sensing molecule. Therefore a synthase is expressed to produce a new AHL signal.<br />
<br />
=== Memorising ===<br />
<br />
Alongside the AHL synthase, GFP is produced according to the XOR output. Th fluorescent protein acts both as long term memory for the colony state and as visual signal, storing and showing the progressive pattern formation.<br />
<br />
=== Sending ===<br />
<br />
The produced AHL diffuses through the channels to the rows below, becoming the input of the following layer. The speed of this directed diffusion determines the time needed from the pattern to emerge. You can find out more information about diffusion and chip design on our [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel modeling page], our [https://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion_On_Chip experimental results page] and our [https://2014.igem.org/Team:ETH_Zurich/lab/chip chip page].<br />
<br />
<br />
<br />
<br />
<br/><br />
<br/><br />
<br/><br />
[[File:ETH_Zurich_Information_Processing_chip.png|center|600px|thumb|'''Figure 2'''The information pathway in the project Mosai''coli'' on chip level. After initializing the signal, it propagates through the wells with directed diffusion. In each well, bacterial colonies have to be able to proceed in the information pathway: detecting (sensing), integrating (computing), producing and sending. These successive iterations leads to possible error propagation. Robustness is one major issue of our system.]]<br />
<br />
<html></article></html><br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/project/infoproTeam:ETH Zurich/project/infopro2014-10-18T02:36:18Z<p>Mbahls: /* The goal: Emergence of patterns via information processing */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Information Processing}}<br />
<html><article></html><br />
<br />
== Information Processing ==<br />
<br />
<br />
=== About the track ===<br />
<br />
<br />
[https://2014.igem.org/Tracks/Information_Processing ''«Information Processing in iGEM covers a diverse range of projects. IP teams are not trying to solve a real world problem with practical applications, but to tackle an interesting problem that might otherwise not attract attention. Teams enter this track if they are attempting projects such as building elements of a biological computer, creating a game using biology or working on a signal processing challenges. Engineering ways to make biological systems perform computation is one of the core goals of synthetic biology. We generally work at the DNA level, engineering systems to function using BioBricks. In most biological systems, protein-protein interactions are where the majority of processing takes place. Being able to design proteins to accomplish computation would allow for systems to function on a much faster timescale than the current transcription-translation paradigm»'' (iGEM mainpage)]<br />
<br />
=== Why we chose this track ===<br />
<br />
With our project, Mosai''coli'', we investigate the emergence of complex patterns from simple mathematical rules. Such rules can be reduced to Boolean logic gates, in our case XOR gates. The computations were implemented with integrases, proteins modifing DNA between specific sites. These modifications in turn influence the expression of other proteins which can then indicate the previous change on the genetic level and chemically wire the information to the next cells, again on the protein or small-molecule level. This wiring of information and repeated information processing allows the construction of cellular automata and eventually biocomputers.<br />
<br />
=== The goal: Emergence of patterns via information processing===<br />
We implement a cellular automaton with bacterial cells containing our logic circuitry. Each bacterial colony serves as a core, computing an XOR gate. First, a sensor device detects the input, ''N''-acyl homoserine lactones (AHL). Then, the cell integrates this signal through a logic gate, performed by serine integrases by sensing on the protein level and acting on the DNA level. A necessary post-processing step allows then the production of a new AHL variant due to activated gene expression through the integrase. Meanwhile, green fluorescent protein (GFP) indicates the state of the colony and serves as a long-lasting visual read out. The produced AHL output-signal then propagates in a directed fashion through a millifluidic grid to the next bacterial colony. This iterative process faces the challenges of leakiness, cross-talk, protein-level computation and exact diffusion steps. This information pathway is shown in figure 1.<br />
<br />
<br />
[[File:ETH_Zurich2014_info_processing1.png|center|800px|thumb|'''Figure 1 The information pathway in the project Mosai''coli'' on a colony level.''' Each cell colony is part of a greater memory unit (left-hand side) and gets its input information from the neighboring colonies. The input is given in the form of two different AHLs, here named A and B. This information is then step-wise processed in each of the colonies: sensing - computing - producing - sending. The output molecules serve then as the input for the next colony (middle and left-hand side). This iterative process allows the information processing from the top row of the memory unit to the bottom by chemical wiring via diffusing signals and biocomputing inside of the colonies.]]<br />
<br />
=== The steps involved: From sensing to sending===<br />
<br />
=== Sensing ===<br />
<br />
Diffused HSL molecules are identified via [https://2014.igem.org/Team:ETH_Zurich/expresults#Quorum_Sensing quorum sensing machinery]. The [https://2014.igem.org/Team:ETH_Zurich/modeling/qs transfer function] between concentration of sensed HSL to promoter activation deviates from the ideal one due to leakiness and crosstalk between HSL molecules, regulatory proteins and promoters (we characterized it for [http://parts.igem.org/Part:BBa_R0062 Lux promoter (BBa_R0062)], [http://parts.igem.org/Part:BBa_R0079 Las promoter (BBa_R0079)] and [http://parts.igem.org/Part:BBa_R0071 Rhl promoter (BBa_R0071)]). To limit the risk of error propagation, we implemented a [http://parts.igem.org/Part:BBa_K1541000 riboregulated system].<br />
<br />
=== Computing ===<br />
<br />
[https://2014.igem.org/Team:ETH_Zurich/expresults#Integrases Integrases] compute an [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR logic gate] via single switching or double switching of a terminator. Their behavior depends on a set of [https://2014.igem.org/Team:ETH_Zurich/modeling/int parameters].<br />
<br />
=== Producing ===<br />
<br />
To propagate the computation results, the output has to be translated again into a quorum sensing molecule. Therefore a synthase is expressed to produce a new AHL signal.<br />
<br />
=== Memorising ===<br />
<br />
Alongside the AHL synthase, GFP is produced according to the XOR output. Th fluorescent protein acts both as long term memory for the colony state and as visual signal, storing and showing the progressive pattern formation.<br />
<br />
=== Sending ===<br />
<br />
The produced AHL diffuses through the channels to the rows below, becoming the input of the following layer. The speed of this directed diffusion determines the time needed from the pattern to emerge. You can find out more information about diffusion and chip design on our [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel modeling page], our [https://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion_On_Chip experimental results page] and our [https://2014.igem.org/Team:ETH_Zurich/lab/chip chip page].<br />
<br />
<br />
<br />
<br />
<br/><br />
<br/><br />
<br/><br />
[[File:ETH_Zurich_Information_Processing_chip.png|center|600px|thumb|'''Figure 2'''The information pathway in the project Mosai''coli'' on chip level. After initializing the signal, it propagates through the wells with directed diffusion. In each well, bacterial colonies have to be able to proceed in the information pathway: detecting (sensing), integrating (computing), producing and sending. These successive iterations leads to possible error propagation. Robustness is one major issue of our system.]]<br />
<br />
<html></article></html><br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/project/infoproTeam:ETH Zurich/project/infopro2014-10-18T02:34:11Z<p>Mbahls: /* The goal: Emergence of patterns via information processing */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Information Processing}}<br />
<html><article></html><br />
<br />
== Information Processing ==<br />
<br />
<br />
=== About the track ===<br />
<br />
<br />
[https://2014.igem.org/Tracks/Information_Processing ''«Information Processing in iGEM covers a diverse range of projects. IP teams are not trying to solve a real world problem with practical applications, but to tackle an interesting problem that might otherwise not attract attention. Teams enter this track if they are attempting projects such as building elements of a biological computer, creating a game using biology or working on a signal processing challenges. Engineering ways to make biological systems perform computation is one of the core goals of synthetic biology. We generally work at the DNA level, engineering systems to function using BioBricks. In most biological systems, protein-protein interactions are where the majority of processing takes place. Being able to design proteins to accomplish computation would allow for systems to function on a much faster timescale than the current transcription-translation paradigm»'' (iGEM mainpage)]<br />
<br />
=== Why we chose this track ===<br />
<br />
With our project, Mosai''coli'', we investigate the emergence of complex patterns from simple mathematical rules. Such rules can be reduced to Boolean logic gates, in our case XOR gates. The computations were implemented with integrases, proteins modifing DNA between specific sites. These modifications in turn influence the expression of other proteins which can then indicate the previous change on the genetic level and chemically wire the information to the next cells, again on the protein or small-molecule level. This wiring of information and repeated information processing allows the construction of cellular automata and eventually biocomputers.<br />
<br />
=== The goal: Emergence of patterns via information processing===<br />
We implement a cellular automaton with bacterial cells containing our logic circuitry. Each bacterial colony serves as a core, computing an XOR gate. First, a sensor device detects the input, ''N''-acyl homoserine lactones (AHL). Then, the cell integrates this signal through a logic gate, performed by serine integrases by sensing on the protein level and acting on the DNA level. A necessary post-processing step allows then the production of a new AHL variant due to activated gene expression through the integrase. Meanwhile, green fluorescent protein (GFP) indicates the state of the colony and serves as a long-lasting visual read out. The produced AHL output-signal then propagates in a directed fashion through a millifluidic grid to the next bacterial colony. This iterative process faces the challenges of leakiness, cross-talk, protein-level computation and exact diffusion steps. This information pathway is shown in figure 1.<br />
<br />
<br />
[[File:ETH_Zurich2014_info_processing1.png|center|800px|thumb|'''Figure 1 The information pathway in the project Mosai''coli'' on a colony level.''' Each cell colony is part of a greater memory unit (left-hand site) and gets its input information from the neighboring colonies. The input is given in the form of two different AHLs, here named A and B. This information is then step-wise processed in each of the colonies: sensing - computing - producing - sending. The output molecules ('sender') serve then as the input for the next colony. This iterative process allows the information processing from the top row of the memory unit to the bottom by chemical wiring of diffusing signals and biocomputing inside of the colonies.]]<br />
<br />
=== The steps involved: From sensing to sending===<br />
<br />
=== Sensing ===<br />
<br />
Diffused HSL molecules are identified via [https://2014.igem.org/Team:ETH_Zurich/expresults#Quorum_Sensing quorum sensing machinery]. The [https://2014.igem.org/Team:ETH_Zurich/modeling/qs transfer function] between concentration of sensed HSL to promoter activation deviates from the ideal one due to leakiness and crosstalk between HSL molecules, regulatory proteins and promoters (we characterized it for [http://parts.igem.org/Part:BBa_R0062 Lux promoter (BBa_R0062)], [http://parts.igem.org/Part:BBa_R0079 Las promoter (BBa_R0079)] and [http://parts.igem.org/Part:BBa_R0071 Rhl promoter (BBa_R0071)]). To limit the risk of error propagation, we implemented a [http://parts.igem.org/Part:BBa_K1541000 riboregulated system].<br />
<br />
=== Computing ===<br />
<br />
[https://2014.igem.org/Team:ETH_Zurich/expresults#Integrases Integrases] compute an [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR logic gate] via single switching or double switching of a terminator. Their behavior depends on a set of [https://2014.igem.org/Team:ETH_Zurich/modeling/int parameters].<br />
<br />
=== Producing ===<br />
<br />
To propagate the computation results, the output has to be translated again into a quorum sensing molecule. Therefore a synthase is expressed to produce a new AHL signal.<br />
<br />
=== Memorising ===<br />
<br />
Alongside the AHL synthase, GFP is produced according to the XOR output. Th fluorescent protein acts both as long term memory for the colony state and as visual signal, storing and showing the progressive pattern formation.<br />
<br />
=== Sending ===<br />
<br />
The produced AHL diffuses through the channels to the rows below, becoming the input of the following layer. The speed of this directed diffusion determines the time needed from the pattern to emerge. You can find out more information about diffusion and chip design on our [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel modeling page], our [https://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion_On_Chip experimental results page] and our [https://2014.igem.org/Team:ETH_Zurich/lab/chip chip page].<br />
<br />
<br />
<br />
<br />
<br/><br />
<br/><br />
<br/><br />
[[File:ETH_Zurich_Information_Processing_chip.png|center|600px|thumb|'''Figure 2'''The information pathway in the project Mosai''coli'' on chip level. After initializing the signal, it propagates through the wells with directed diffusion. In each well, bacterial colonies have to be able to proceed in the information pathway: detecting (sensing), integrating (computing), producing and sending. These successive iterations leads to possible error propagation. Robustness is one major issue of our system.]]<br />
<br />
<html></article></html><br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/project/infoproTeam:ETH Zurich/project/infopro2014-10-18T02:33:51Z<p>Mbahls: /* The goal: Emergence of patterns via information processing */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Information Processing}}<br />
<html><article></html><br />
<br />
== Information Processing ==<br />
<br />
<br />
=== About the track ===<br />
<br />
<br />
[https://2014.igem.org/Tracks/Information_Processing ''«Information Processing in iGEM covers a diverse range of projects. IP teams are not trying to solve a real world problem with practical applications, but to tackle an interesting problem that might otherwise not attract attention. Teams enter this track if they are attempting projects such as building elements of a biological computer, creating a game using biology or working on a signal processing challenges. Engineering ways to make biological systems perform computation is one of the core goals of synthetic biology. We generally work at the DNA level, engineering systems to function using BioBricks. In most biological systems, protein-protein interactions are where the majority of processing takes place. Being able to design proteins to accomplish computation would allow for systems to function on a much faster timescale than the current transcription-translation paradigm»'' (iGEM mainpage)]<br />
<br />
=== Why we chose this track ===<br />
<br />
With our project, Mosai''coli'', we investigate the emergence of complex patterns from simple mathematical rules. Such rules can be reduced to Boolean logic gates, in our case XOR gates. The computations were implemented with integrases, proteins modifing DNA between specific sites. These modifications in turn influence the expression of other proteins which can then indicate the previous change on the genetic level and chemically wire the information to the next cells, again on the protein or small-molecule level. This wiring of information and repeated information processing allows the construction of cellular automata and eventually biocomputers.<br />
<br />
=== The goal: Emergence of patterns via information processing===<br />
We implement a cellular automaton with bacterial cells containing our logic circuitry. Each bacterial colony serves as a core, computing an XOR gate. First, a sensor device detects the input, ''N''-acyl homoserine lactones (AHL). Then, the cell integrates this signal through a logic gate, performed by serine integrases by sensing on the protein level and acting on the DNA level. A necessary post-processing step allows then the production of a new AHL variant due to activated gene expression through the integrase. Meanwhile, green fluorescent protein (GFP) indicates the state of the colony and serves as a long-lasting visual read out. The produced AHL output-signal then propagates in a directed fashion through a millifluidic grid to the next bacterial colony. This iterative process faces the challenges of leakiness, cross-talk, protein-level computation and exact diffusion steps. this information pathway is shown in figure 1.<br />
<br />
<br />
[[File:ETH_Zurich2014_info_processing1.png|center|800px|thumb|'''Figure 1 The information pathway in the project Mosai''coli'' on a colony level.''' Each cell colony is part of a greater memory unit (left-hand site) and gets its input information from the neighboring colonies. The input is given in the form of two different AHLs, here named A and B. This information is then step-wise processed in each of the colonies: sensing - computing - producing - sending. The output molecules ('sender') serve then as the input for the next colony. This iterative process allows the information processing from the top row of the memory unit to the bottom by chemical wiring of diffusing signals and biocomputing inside of the colonies.]]<br />
<br />
=== The steps involved: From sensing to sending===<br />
<br />
=== Sensing ===<br />
<br />
Diffused HSL molecules are identified via [https://2014.igem.org/Team:ETH_Zurich/expresults#Quorum_Sensing quorum sensing machinery]. The [https://2014.igem.org/Team:ETH_Zurich/modeling/qs transfer function] between concentration of sensed HSL to promoter activation deviates from the ideal one due to leakiness and crosstalk between HSL molecules, regulatory proteins and promoters (we characterized it for [http://parts.igem.org/Part:BBa_R0062 Lux promoter (BBa_R0062)], [http://parts.igem.org/Part:BBa_R0079 Las promoter (BBa_R0079)] and [http://parts.igem.org/Part:BBa_R0071 Rhl promoter (BBa_R0071)]). To limit the risk of error propagation, we implemented a [http://parts.igem.org/Part:BBa_K1541000 riboregulated system].<br />
<br />
=== Computing ===<br />
<br />
[https://2014.igem.org/Team:ETH_Zurich/expresults#Integrases Integrases] compute an [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR logic gate] via single switching or double switching of a terminator. Their behavior depends on a set of [https://2014.igem.org/Team:ETH_Zurich/modeling/int parameters].<br />
<br />
=== Producing ===<br />
<br />
To propagate the computation results, the output has to be translated again into a quorum sensing molecule. Therefore a synthase is expressed to produce a new AHL signal.<br />
<br />
=== Memorising ===<br />
<br />
Alongside the AHL synthase, GFP is produced according to the XOR output. Th fluorescent protein acts both as long term memory for the colony state and as visual signal, storing and showing the progressive pattern formation.<br />
<br />
=== Sending ===<br />
<br />
The produced AHL diffuses through the channels to the rows below, becoming the input of the following layer. The speed of this directed diffusion determines the time needed from the pattern to emerge. You can find out more information about diffusion and chip design on our [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel modeling page], our [https://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion_On_Chip experimental results page] and our [https://2014.igem.org/Team:ETH_Zurich/lab/chip chip page].<br />
<br />
<br />
<br />
<br />
<br/><br />
<br/><br />
<br/><br />
[[File:ETH_Zurich_Information_Processing_chip.png|center|600px|thumb|'''Figure 2'''The information pathway in the project Mosai''coli'' on chip level. After initializing the signal, it propagates through the wells with directed diffusion. In each well, bacterial colonies have to be able to proceed in the information pathway: detecting (sensing), integrating (computing), producing and sending. These successive iterations leads to possible error propagation. Robustness is one major issue of our system.]]<br />
<br />
<html></article></html><br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/project/infoproTeam:ETH Zurich/project/infopro2014-10-18T02:22:26Z<p>Mbahls: /* The goal: Emergence of patterns via information processing */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Information Processing}}<br />
<html><article></html><br />
<br />
== Information Processing ==<br />
<br />
<br />
=== About the track ===<br />
<br />
<br />
[https://2014.igem.org/Tracks/Information_Processing ''«Information Processing in iGEM covers a diverse range of projects. IP teams are not trying to solve a real world problem with practical applications, but to tackle an interesting problem that might otherwise not attract attention. Teams enter this track if they are attempting projects such as building elements of a biological computer, creating a game using biology or working on a signal processing challenges. Engineering ways to make biological systems perform computation is one of the core goals of synthetic biology. We generally work at the DNA level, engineering systems to function using BioBricks. In most biological systems, protein-protein interactions are where the majority of processing takes place. Being able to design proteins to accomplish computation would allow for systems to function on a much faster timescale than the current transcription-translation paradigm»'' (iGEM mainpage)]<br />
<br />
=== Why we chose this track ===<br />
<br />
With our project, Mosai''coli'', we investigate the emergence of complex patterns from simple mathematical rules. Such rules can be reduced to Boolean logic gates, in our case XOR gates. The computations were implemented with integrases, proteins modifing DNA between specific sites. These modifications in turn influence the expression of other proteins which can then indicate the previous change on the genetic level and chemically wire the information to the next cells, again on the protein or small-molecule level. This wiring of information and repeated information processing allows the construction of cellular automata and eventually biocomputers.<br />
<br />
=== The goal: Emergence of patterns via information processing===<br />
We implement a cellular automaton with bacterial cells containing our logic circuitry. Each bacterial colony serves as a core, computing an XOR gate. First, a sensor device detects the input, ''N''-acyl homoserine lactones (AHL). Then, the cell integrates this signal through a logic gate, performed by serine integrases by sensing on the protein level and acting on the DNA level. A necessary post-processing step allows then the production of a new AHL variant due to activated gene expression through the integrase. Meanwhile, green fluorescent protein (GFP) indicates the state of the colony and serves as a long-lasting visual read out. The produced AHL output-signal then propagates in a directed fashion through a millifluidic grid to the next bacterial colony. This iterative process faces the challenges of leakiness, cross-talk, protein-level computation and exact diffusion steps. this information pathway is shown in figure 1.<br />
<br />
<br />
[[File:ETH_Zurich2014_info_processing1.png|center|800px|thumb|'''Figure 1''' The information pathway in the project Mosai''coli'' on a colony level.]]<br />
<br />
=== The steps involved: From sensing to sending===<br />
<br />
=== Sensing ===<br />
<br />
Diffused HSL molecules are identified via [https://2014.igem.org/Team:ETH_Zurich/expresults#Quorum_Sensing quorum sensing machinery]. The [https://2014.igem.org/Team:ETH_Zurich/modeling/qs transfer function] between concentration of sensed HSL to promoter activation deviates from the ideal one due to leakiness and crosstalk between HSL molecules, regulatory proteins and promoters (we characterized it for [http://parts.igem.org/Part:BBa_R0062 Lux promoter (BBa_R0062)], [http://parts.igem.org/Part:BBa_R0079 Las promoter (BBa_R0079)] and [http://parts.igem.org/Part:BBa_R0071 Rhl promoter (BBa_R0071)]). To limit the risk of error propagation, we implemented a [http://parts.igem.org/Part:BBa_K1541000 riboregulated system].<br />
<br />
=== Computing ===<br />
<br />
[https://2014.igem.org/Team:ETH_Zurich/expresults#Integrases Integrases] compute an [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR logic gate] via single switching or double switching of a terminator. Their behavior depends on a set of [https://2014.igem.org/Team:ETH_Zurich/modeling/int parameters].<br />
<br />
=== Producing ===<br />
<br />
To propagate the computation results, the output has to be translated again into a quorum sensing molecule. Therefore a synthase is expressed to produce a new AHL signal.<br />
<br />
=== Memorising ===<br />
<br />
Alongside the AHL synthase, GFP is produced according to the XOR output. Th fluorescent protein acts both as long term memory for the colony state and as visual signal, storing and showing the progressive pattern formation.<br />
<br />
=== Sending ===<br />
<br />
The produced AHL diffuses through the channels to the rows below, becoming the input of the following layer. The speed of this directed diffusion determines the time needed from the pattern to emerge. You can find out more information about diffusion and chip design on our [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel modeling page], our [https://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion_On_Chip experimental results page] and our [https://2014.igem.org/Team:ETH_Zurich/lab/chip chip page].<br />
<br />
<br />
<br />
<br />
<br/><br />
<br/><br />
<br/><br />
[[File:ETH_Zurich_Information_Processing_chip.png|center|600px|thumb|'''Figure 2'''The information pathway in the project Mosai''coli'' on chip level. After initializing the signal, it propagates through the wells with directed diffusion. In each well, bacterial colonies have to be able to proceed in the information pathway: detecting (sensing), integrating (computing), producing and sending. These successive iterations leads to possible error propagation. Robustness is one major issue of our system.]]<br />
<br />
<html></article></html><br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/project/infoproTeam:ETH Zurich/project/infopro2014-10-18T02:21:54Z<p>Mbahls: /* The goal: Emergence of patterns via information processing */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Information Processing}}<br />
<html><article></html><br />
<br />
== Information Processing ==<br />
<br />
<br />
=== About the track ===<br />
<br />
<br />
[https://2014.igem.org/Tracks/Information_Processing ''«Information Processing in iGEM covers a diverse range of projects. IP teams are not trying to solve a real world problem with practical applications, but to tackle an interesting problem that might otherwise not attract attention. Teams enter this track if they are attempting projects such as building elements of a biological computer, creating a game using biology or working on a signal processing challenges. Engineering ways to make biological systems perform computation is one of the core goals of synthetic biology. We generally work at the DNA level, engineering systems to function using BioBricks. In most biological systems, protein-protein interactions are where the majority of processing takes place. Being able to design proteins to accomplish computation would allow for systems to function on a much faster timescale than the current transcription-translation paradigm»'' (iGEM mainpage)]<br />
<br />
=== Why we chose this track ===<br />
<br />
With our project, Mosai''coli'', we investigate the emergence of complex patterns from simple mathematical rules. Such rules can be reduced to Boolean logic gates, in our case XOR gates. The computations were implemented with integrases, proteins modifing DNA between specific sites. These modifications in turn influence the expression of other proteins which can then indicate the previous change on the genetic level and chemically wire the information to the next cells, again on the protein or small-molecule level. This wiring of information and repeated information processing allows the construction of cellular automata and eventually biocomputers.<br />
<br />
=== The goal: Emergence of patterns via information processing===<br />
We implement a cellular automaton with bacterial cells containing our logic circuitry. Each bacterial colony serves as a core, computing an XOR gate. First, a sensor device detects the input, ''N''-acyl homoserine lactones (AHL). Then, the cell integrates this signal through a logic gate, performed by serine integrases by sensing on the protein level and acting on the DNA level. A necessary post-processing step allows then the production of a new AHL variant due to activated gene expression through the integrase. Meanwhile, green fluorescent protein (GFP) indicates the state of the colony and serves as a long-lasting visual read out. The produced AHL output-signal then propagates in a directed fashion through a millifluidic grid to the next bacterial colony. This iterative process faces the challenges of leakiness, cross-talk, protein-level computation and exact diffusion steps.<br />
<br />
<br />
[[File:ETH_Zurich2014_info_processing1.png|center|800px|thumb|'''Figure 1''' The information pathway in the project Mosai''coli'' on a colony level.]]<br />
<br />
=== The steps involved: From sensing to sending===<br />
<br />
=== Sensing ===<br />
<br />
Diffused HSL molecules are identified via [https://2014.igem.org/Team:ETH_Zurich/expresults#Quorum_Sensing quorum sensing machinery]. The [https://2014.igem.org/Team:ETH_Zurich/modeling/qs transfer function] between concentration of sensed HSL to promoter activation deviates from the ideal one due to leakiness and crosstalk between HSL molecules, regulatory proteins and promoters (we characterized it for [http://parts.igem.org/Part:BBa_R0062 Lux promoter (BBa_R0062)], [http://parts.igem.org/Part:BBa_R0079 Las promoter (BBa_R0079)] and [http://parts.igem.org/Part:BBa_R0071 Rhl promoter (BBa_R0071)]). To limit the risk of error propagation, we implemented a [http://parts.igem.org/Part:BBa_K1541000 riboregulated system].<br />
<br />
=== Computing ===<br />
<br />
[https://2014.igem.org/Team:ETH_Zurich/expresults#Integrases Integrases] compute an [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR logic gate] via single switching or double switching of a terminator. Their behavior depends on a set of [https://2014.igem.org/Team:ETH_Zurich/modeling/int parameters].<br />
<br />
=== Producing ===<br />
<br />
To propagate the computation results, the output has to be translated again into a quorum sensing molecule. Therefore a synthase is expressed to produce a new AHL signal.<br />
<br />
=== Memorising ===<br />
<br />
Alongside the AHL synthase, GFP is produced according to the XOR output. Th fluorescent protein acts both as long term memory for the colony state and as visual signal, storing and showing the progressive pattern formation.<br />
<br />
=== Sending ===<br />
<br />
The produced AHL diffuses through the channels to the rows below, becoming the input of the following layer. The speed of this directed diffusion determines the time needed from the pattern to emerge. You can find out more information about diffusion and chip design on our [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel modeling page], our [https://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion_On_Chip experimental results page] and our [https://2014.igem.org/Team:ETH_Zurich/lab/chip chip page].<br />
<br />
<br />
<br />
<br />
<br/><br />
<br/><br />
<br/><br />
[[File:ETH_Zurich_Information_Processing_chip.png|center|600px|thumb|'''Figure 2'''The information pathway in the project Mosai''coli'' on chip level. After initializing the signal, it propagates through the wells with directed diffusion. In each well, bacterial colonies have to be able to proceed in the information pathway: detecting (sensing), integrating (computing), producing and sending. These successive iterations leads to possible error propagation. Robustness is one major issue of our system.]]<br />
<br />
<html></article></html><br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/project/infoproTeam:ETH Zurich/project/infopro2014-10-18T02:21:18Z<p>Mbahls: /* The goal: Emergence of patterns via information processing */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Information Processing}}<br />
<html><article></html><br />
<br />
== Information Processing ==<br />
<br />
<br />
=== About the track ===<br />
<br />
<br />
[https://2014.igem.org/Tracks/Information_Processing ''«Information Processing in iGEM covers a diverse range of projects. IP teams are not trying to solve a real world problem with practical applications, but to tackle an interesting problem that might otherwise not attract attention. Teams enter this track if they are attempting projects such as building elements of a biological computer, creating a game using biology or working on a signal processing challenges. Engineering ways to make biological systems perform computation is one of the core goals of synthetic biology. We generally work at the DNA level, engineering systems to function using BioBricks. In most biological systems, protein-protein interactions are where the majority of processing takes place. Being able to design proteins to accomplish computation would allow for systems to function on a much faster timescale than the current transcription-translation paradigm»'' (iGEM mainpage)]<br />
<br />
=== Why we chose this track ===<br />
<br />
With our project, Mosai''coli'', we investigate the emergence of complex patterns from simple mathematical rules. Such rules can be reduced to Boolean logic gates, in our case XOR gates. The computations were implemented with integrases, proteins modifing DNA between specific sites. These modifications in turn influence the expression of other proteins which can then indicate the previous change on the genetic level and chemically wire the information to the next cells, again on the protein or small-molecule level. This wiring of information and repeated information processing allows the construction of cellular automata and eventually biocomputers.<br />
<br />
=== The goal: Emergence of patterns via information processing===<br />
We implement a cellular automaton with bacterial cells containing our logic circuitry. Each bacterial colony serves as a core, computing an XOR gate. First, a sensor device detects the input, ''N''-acyl homoserine lactones (AHL). Then, the cell integrates this signal through a logic gate, performed by serine integrases by sensing on the protein level and acting on the DNA level. A necessary post-processing step allows then the production of a new AHL variant due to activated gene expression through the integrase. Meanwhile, green fluorescent protein (GFP) indicates the state of the colony and serves as a long-lasting visual read out. The produced AHL output-signal then propagates in a directed fashion through a millifluidic grid to the next bacterial colony. This iterative process faces the challenges of leakiness, cross-talk, protein-level computation and exact diffusion steps.<br />
<br />
<br />
[[File:ETH_Zurich2014_info_processing1.png|center|800px|thumb|'''Figure 1'''The information pathway in the project Mosai''coli'' on a colony level.]]<br />
<br />
=== The steps involved: From sensing to sending===<br />
<br />
=== Sensing ===<br />
<br />
Diffused HSL molecules are identified via [https://2014.igem.org/Team:ETH_Zurich/expresults#Quorum_Sensing quorum sensing machinery]. The [https://2014.igem.org/Team:ETH_Zurich/modeling/qs transfer function] between concentration of sensed HSL to promoter activation deviates from the ideal one due to leakiness and crosstalk between HSL molecules, regulatory proteins and promoters (we characterized it for [http://parts.igem.org/Part:BBa_R0062 Lux promoter (BBa_R0062)], [http://parts.igem.org/Part:BBa_R0079 Las promoter (BBa_R0079)] and [http://parts.igem.org/Part:BBa_R0071 Rhl promoter (BBa_R0071)]). To limit the risk of error propagation, we implemented a [http://parts.igem.org/Part:BBa_K1541000 riboregulated system].<br />
<br />
=== Computing ===<br />
<br />
[https://2014.igem.org/Team:ETH_Zurich/expresults#Integrases Integrases] compute an [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR logic gate] via single switching or double switching of a terminator. Their behavior depends on a set of [https://2014.igem.org/Team:ETH_Zurich/modeling/int parameters].<br />
<br />
=== Producing ===<br />
<br />
To propagate the computation results, the output has to be translated again into a quorum sensing molecule. Therefore a synthase is expressed to produce a new AHL signal.<br />
<br />
=== Memorising ===<br />
<br />
Alongside the AHL synthase, GFP is produced according to the XOR output. Th fluorescent protein acts both as long term memory for the colony state and as visual signal, storing and showing the progressive pattern formation.<br />
<br />
=== Sending ===<br />
<br />
The produced AHL diffuses through the channels to the rows below, becoming the input of the following layer. The speed of this directed diffusion determines the time needed from the pattern to emerge. You can find out more information about diffusion and chip design on our [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel modeling page], our [https://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion_On_Chip experimental results page] and our [https://2014.igem.org/Team:ETH_Zurich/lab/chip chip page].<br />
<br />
<br />
<br />
<br />
<br/><br />
<br/><br />
<br/><br />
[[File:ETH_Zurich_Information_Processing_chip.png|center|600px|thumb|'''Figure 2'''The information pathway in the project Mosai''coli'' on chip level. After initializing the signal, it propagates through the wells with directed diffusion. In each well, bacterial colonies have to be able to proceed in the information pathway: detecting (sensing), integrating (computing), producing and sending. These successive iterations leads to possible error propagation. Robustness is one major issue of our system.]]<br />
<br />
<html></article></html><br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/project/infoproTeam:ETH Zurich/project/infopro2014-10-18T02:18:34Z<p>Mbahls: /* Why we chose this track */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Information Processing}}<br />
<html><article></html><br />
<br />
== Information Processing ==<br />
<br />
<br />
=== About the track ===<br />
<br />
<br />
[https://2014.igem.org/Tracks/Information_Processing ''«Information Processing in iGEM covers a diverse range of projects. IP teams are not trying to solve a real world problem with practical applications, but to tackle an interesting problem that might otherwise not attract attention. Teams enter this track if they are attempting projects such as building elements of a biological computer, creating a game using biology or working on a signal processing challenges. Engineering ways to make biological systems perform computation is one of the core goals of synthetic biology. We generally work at the DNA level, engineering systems to function using BioBricks. In most biological systems, protein-protein interactions are where the majority of processing takes place. Being able to design proteins to accomplish computation would allow for systems to function on a much faster timescale than the current transcription-translation paradigm»'' (iGEM mainpage)]<br />
<br />
=== Why we chose this track ===<br />
<br />
With our project, Mosai''coli'', we investigate the emergence of complex patterns from simple mathematical rules. Such rules can be reduced to Boolean logic gates, in our case XOR gates. The computations were implemented with integrases, proteins modifing DNA between specific sites. These modifications in turn influence the expression of other proteins which can then indicate the previous change on the genetic level and chemically wire the information to the next cells, again on the protein or small-molecule level. This wiring of information and repeated information processing allows the construction of cellular automata and eventually biocomputers.<br />
<br />
=== The goal: Emergence of patterns via information processing===<br />
We implement a cellular automaton with bacterial cells containing our logic circuitry. Each bacterial colony serves as a core, computing an XOR gate. First, a sensor device detects the input, ''N''-acyl homoserine lactones (AHL). Then, the cell integrates this signal through a logic gate, performed by serine integrases on the protein level. A necessary post-processing step allows the production of new AHL variant. Meanwhile, green fluorescent protein (GFP) indicates the state of the colony and serves as a long-lasting visual read out. The produced AHL output-signal then propagates in a directed fashion through a millifluidic grid to the next bacterial colony. This iterative process faces the challenges of leakiness, cross-talk, protein-level computation and exact diffusion steps.<br />
<br />
<br />
[[File:ETH_Zurich2014_info_processing1.png|center|800px|thumb|'''Figure 1'''The information pathway in the project Mosai''coli'' on a colony level.]]<br />
<br />
=== The steps involved: From sensing to sending===<br />
<br />
=== Sensing ===<br />
<br />
Diffused HSL molecules are identified via [https://2014.igem.org/Team:ETH_Zurich/expresults#Quorum_Sensing quorum sensing machinery]. The [https://2014.igem.org/Team:ETH_Zurich/modeling/qs transfer function] between concentration of sensed HSL to promoter activation deviates from the ideal one due to leakiness and crosstalk between HSL molecules, regulatory proteins and promoters (we characterized it for [http://parts.igem.org/Part:BBa_R0062 Lux promoter (BBa_R0062)], [http://parts.igem.org/Part:BBa_R0079 Las promoter (BBa_R0079)] and [http://parts.igem.org/Part:BBa_R0071 Rhl promoter (BBa_R0071)]). To limit the risk of error propagation, we implemented a [http://parts.igem.org/Part:BBa_K1541000 riboregulated system].<br />
<br />
=== Computing ===<br />
<br />
[https://2014.igem.org/Team:ETH_Zurich/expresults#Integrases Integrases] compute an [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR logic gate] via single switching or double switching of a terminator. Their behavior depends on a set of [https://2014.igem.org/Team:ETH_Zurich/modeling/int parameters].<br />
<br />
=== Producing ===<br />
<br />
To propagate the computation results, the output has to be translated again into a quorum sensing molecule. Therefore a synthase is expressed to produce a new AHL signal.<br />
<br />
=== Memorising ===<br />
<br />
Alongside the AHL synthase, GFP is produced according to the XOR output. Th fluorescent protein acts both as long term memory for the colony state and as visual signal, storing and showing the progressive pattern formation.<br />
<br />
=== Sending ===<br />
<br />
The produced AHL diffuses through the channels to the rows below, becoming the input of the following layer. The speed of this directed diffusion determines the time needed from the pattern to emerge. You can find out more information about diffusion and chip design on our [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel modeling page], our [https://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion_On_Chip experimental results page] and our [https://2014.igem.org/Team:ETH_Zurich/lab/chip chip page].<br />
<br />
<br />
<br />
<br />
<br/><br />
<br/><br />
<br/><br />
[[File:ETH_Zurich_Information_Processing_chip.png|center|600px|thumb|'''Figure 2'''The information pathway in the project Mosai''coli'' on chip level. After initializing the signal, it propagates through the wells with directed diffusion. In each well, bacterial colonies have to be able to proceed in the information pathway: detecting (sensing), integrating (computing), producing and sending. These successive iterations leads to possible error propagation. Robustness is one major issue of our system.]]<br />
<br />
<html></article></html><br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/project/infoproTeam:ETH Zurich/project/infopro2014-10-18T02:17:37Z<p>Mbahls: /* The goal: Emergence of patterns via information processing */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Information Processing}}<br />
<html><article></html><br />
<br />
== Information Processing ==<br />
<br />
<br />
=== About the track ===<br />
<br />
<br />
[https://2014.igem.org/Tracks/Information_Processing ''«Information Processing in iGEM covers a diverse range of projects. IP teams are not trying to solve a real world problem with practical applications, but to tackle an interesting problem that might otherwise not attract attention. Teams enter this track if they are attempting projects such as building elements of a biological computer, creating a game using biology or working on a signal processing challenges. Engineering ways to make biological systems perform computation is one of the core goals of synthetic biology. We generally work at the DNA level, engineering systems to function using BioBricks. In most biological systems, protein-protein interactions are where the majority of processing takes place. Being able to design proteins to accomplish computation would allow for systems to function on a much faster timescale than the current transcription-translation paradigm»'' (iGEM mainpage)]<br />
<br />
=== Why we chose this track ===<br />
<br />
With our project, Mosai''coli'', we investigate the emergence of complex patterns from simple mathematical rules. Such rules can be reduced to Boolean logic gates, in our case XOR gates. The computations were implemented with integrases, proteins modifing DNA between specific sites. These modifications in turn influence the expression of other proteins which can then indicate the previous change on the genetic level and chemically wire the information to the next cells, again on the protein level. This wiring of information and repeated information processing allows the construction of cellular automata and eventually biocomputers.<br />
<br />
=== The goal: Emergence of patterns via information processing===<br />
We implement a cellular automaton with bacterial cells containing our logic circuitry. Each bacterial colony serves as a core, computing an XOR gate. First, a sensor device detects the input, ''N''-acyl homoserine lactones (AHL). Then, the cell integrates this signal through a logic gate, performed by serine integrases on the protein level. A necessary post-processing step allows the production of new AHL variant. Meanwhile, green fluorescent protein (GFP) indicates the state of the colony and serves as a long-lasting visual read out. The produced AHL output-signal then propagates in a directed fashion through a millifluidic grid to the next bacterial colony. This iterative process faces the challenges of leakiness, cross-talk, protein-level computation and exact diffusion steps.<br />
<br />
<br />
[[File:ETH_Zurich2014_info_processing1.png|center|800px|thumb|'''Figure 1'''The information pathway in the project Mosai''coli'' on a colony level.]]<br />
<br />
=== The steps involved: From sensing to sending===<br />
<br />
=== Sensing ===<br />
<br />
Diffused HSL molecules are identified via [https://2014.igem.org/Team:ETH_Zurich/expresults#Quorum_Sensing quorum sensing machinery]. The [https://2014.igem.org/Team:ETH_Zurich/modeling/qs transfer function] between concentration of sensed HSL to promoter activation deviates from the ideal one due to leakiness and crosstalk between HSL molecules, regulatory proteins and promoters (we characterized it for [http://parts.igem.org/Part:BBa_R0062 Lux promoter (BBa_R0062)], [http://parts.igem.org/Part:BBa_R0079 Las promoter (BBa_R0079)] and [http://parts.igem.org/Part:BBa_R0071 Rhl promoter (BBa_R0071)]). To limit the risk of error propagation, we implemented a [http://parts.igem.org/Part:BBa_K1541000 riboregulated system].<br />
<br />
=== Computing ===<br />
<br />
[https://2014.igem.org/Team:ETH_Zurich/expresults#Integrases Integrases] compute an [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR logic gate] via single switching or double switching of a terminator. Their behavior depends on a set of [https://2014.igem.org/Team:ETH_Zurich/modeling/int parameters].<br />
<br />
=== Producing ===<br />
<br />
To propagate the computation results, the output has to be translated again into a quorum sensing molecule. Therefore a synthase is expressed to produce a new AHL signal.<br />
<br />
=== Memorising ===<br />
<br />
Alongside the AHL synthase, GFP is produced according to the XOR output. Th fluorescent protein acts both as long term memory for the colony state and as visual signal, storing and showing the progressive pattern formation.<br />
<br />
=== Sending ===<br />
<br />
The produced AHL diffuses through the channels to the rows below, becoming the input of the following layer. The speed of this directed diffusion determines the time needed from the pattern to emerge. You can find out more information about diffusion and chip design on our [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel modeling page], our [https://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion_On_Chip experimental results page] and our [https://2014.igem.org/Team:ETH_Zurich/lab/chip chip page].<br />
<br />
<br />
<br />
<br />
<br/><br />
<br/><br />
<br/><br />
[[File:ETH_Zurich_Information_Processing_chip.png|center|600px|thumb|'''Figure 2'''The information pathway in the project Mosai''coli'' on chip level. After initializing the signal, it propagates through the wells with directed diffusion. In each well, bacterial colonies have to be able to proceed in the information pathway: detecting (sensing), integrating (computing), producing and sending. These successive iterations leads to possible error propagation. Robustness is one major issue of our system.]]<br />
<br />
<html></article></html><br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/project/infoproTeam:ETH Zurich/project/infopro2014-10-18T02:08:23Z<p>Mbahls: /* The goal: Pattern emergence via information processing */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Information Processing}}<br />
<html><article></html><br />
<br />
== Information Processing ==<br />
<br />
<br />
=== About the track ===<br />
<br />
<br />
[https://2014.igem.org/Tracks/Information_Processing ''«Information Processing in iGEM covers a diverse range of projects. IP teams are not trying to solve a real world problem with practical applications, but to tackle an interesting problem that might otherwise not attract attention. Teams enter this track if they are attempting projects such as building elements of a biological computer, creating a game using biology or working on a signal processing challenges. Engineering ways to make biological systems perform computation is one of the core goals of synthetic biology. We generally work at the DNA level, engineering systems to function using BioBricks. In most biological systems, protein-protein interactions are where the majority of processing takes place. Being able to design proteins to accomplish computation would allow for systems to function on a much faster timescale than the current transcription-translation paradigm»'' (iGEM mainpage)]<br />
<br />
=== Why we chose this track ===<br />
<br />
With our project, Mosai''coli'', we investigate the emergence of complex patterns from simple mathematical rules. Such rules can be reduced to Boolean logic gates, in our case XOR gates. The computations were implemented with integrases, proteins modifing DNA between specific sites. These modifications in turn influence the expression of other proteins which can then indicate the previous change on the genetic level and chemically wire the information to the next cells, again on the protein level. This wiring of information and repeated information processing allows the construction of cellular automata and eventually biocomputers.<br />
<br />
=== The goal: Emergence of patterns via information processing===<br />
We implement a cellular automaton in bacterial colonies. Each bacterial colony is a core, computing an XOR gate. A sensor device detects the inputs, HSL molecules. Then, the cell integrates the signal through a logic gate, performed by proteins, the serine integrases. A necessary post processing step generates the production of HSL molecules. Meanwhile, GFP, a visual read out, longlastingly indicates the state of the well. The produced signal then propagates in a directive fashion through a millifluidic chip. This iterative process faces the challenges of leakiness, cross-talk, protein-level computation and diffusion.<br />
<br/><br />
<br/><br />
<br />
[[File:ETH_Zurich2014_info_processing1.png|center|800px|thumb|'''Figure 1'''The information pathway in the project Mosai''coli'' on a colony level.]]<br />
<br />
=== The steps involved: From sensing to sending===<br />
<br />
=== Sensing ===<br />
<br />
Diffused HSL molecules are identified via [https://2014.igem.org/Team:ETH_Zurich/expresults#Quorum_Sensing quorum sensing machinery]. The [https://2014.igem.org/Team:ETH_Zurich/modeling/qs transfer function] between concentration of sensed HSL to promoter activation deviates from the ideal one due to leakiness and crosstalk between HSL molecules, regulatory proteins and promoters (we characterized it for [http://parts.igem.org/Part:BBa_R0062 Lux promoter (BBa_R0062)], [http://parts.igem.org/Part:BBa_R0079 Las promoter (BBa_R0079)] and [http://parts.igem.org/Part:BBa_R0071 Rhl promoter (BBa_R0071)]). To limit the risk of error propagation, we implemented a [http://parts.igem.org/Part:BBa_K1541000 riboregulated system].<br />
<br />
=== Computing ===<br />
<br />
[https://2014.igem.org/Team:ETH_Zurich/expresults#Integrases Integrases] compute an [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR logic gate] via single switching or double switching of a terminator. Their behavior depends on a set of [https://2014.igem.org/Team:ETH_Zurich/modeling/int parameters].<br />
<br />
=== Producing ===<br />
<br />
To propagate the computation results, the output has to be translated again into a quorum sensing molecule. Therefore a synthase is expressed to produce a new AHL signal.<br />
<br />
=== Memorising ===<br />
<br />
Alongside the AHL synthase, GFP is produced according to the XOR output. Th fluorescent protein acts both as long term memory for the colony state and as visual signal, storing and showing the progressive pattern formation.<br />
<br />
=== Sending ===<br />
<br />
The produced AHL diffuses through the channels to the rows below, becoming the input of the following layer. The speed of this directed diffusion determines the time needed from the pattern to emerge. You can find out more information about diffusion and chip design on our [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel modeling page], our [https://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion_On_Chip experimental results page] and our [https://2014.igem.org/Team:ETH_Zurich/lab/chip chip page].<br />
<br />
<br />
<br />
<br />
<br/><br />
<br/><br />
<br/><br />
[[File:ETH_Zurich_Information_Processing_chip.png|center|600px|thumb|'''Figure 2'''The information pathway in the project Mosai''coli'' on chip level. After initializing the signal, it propagates through the wells with directed diffusion. In each well, bacterial colonies have to be able to proceed in the information pathway: detecting (sensing), integrating (computing), producing and sending. These successive iterations leads to possible error propagation. Robustness is one major issue of our system.]]<br />
<br />
<html></article></html><br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/project/infoproTeam:ETH Zurich/project/infopro2014-10-18T02:07:35Z<p>Mbahls: /* Why we chose this track */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Information Processing}}<br />
<html><article></html><br />
<br />
== Information Processing ==<br />
<br />
<br />
=== About the track ===<br />
<br />
<br />
[https://2014.igem.org/Tracks/Information_Processing ''«Information Processing in iGEM covers a diverse range of projects. IP teams are not trying to solve a real world problem with practical applications, but to tackle an interesting problem that might otherwise not attract attention. Teams enter this track if they are attempting projects such as building elements of a biological computer, creating a game using biology or working on a signal processing challenges. Engineering ways to make biological systems perform computation is one of the core goals of synthetic biology. We generally work at the DNA level, engineering systems to function using BioBricks. In most biological systems, protein-protein interactions are where the majority of processing takes place. Being able to design proteins to accomplish computation would allow for systems to function on a much faster timescale than the current transcription-translation paradigm»'' (iGEM mainpage)]<br />
<br />
=== Why we chose this track ===<br />
<br />
With our project, Mosai''coli'', we investigate the emergence of complex patterns from simple mathematical rules. Such rules can be reduced to Boolean logic gates, in our case XOR gates. The computations were implemented with integrases, proteins modifing DNA between specific sites. These modifications in turn influence the expression of other proteins which can then indicate the previous change on the genetic level and chemically wire the information to the next cells, again on the protein level. This wiring of information and repeated information processing allows the construction of cellular automata and eventually biocomputers.<br />
<br />
=== The goal: Pattern emergence via information processing===<br />
We implement a cellular automaton in bacterial colonies. Each bacterial colony is a core, computing an XOR gate. A sensor device detects the inputs, HSL molecules. Then, the cell integrates the signal through a logic gate, performed by proteins, the serine integrases. A necessary post processing step generates the production of HSL molecules. Meanwhile, GFP, a visual read out, longlastingly indicates the state of the well. The produced signal then propagates in a directive fashion through a millifluidic chip. This iterative process faces the challenges of leakiness, cross-talk, protein-level computation and diffusion.<br />
<br/><br />
<br/><br />
<br />
[[File:ETH_Zurich2014_info_processing1.png|center|800px|thumb|'''Figure 1'''The information pathway in the project Mosai''coli'' on a colony level.]]<br />
<br />
<br />
=== The steps involved: From sensing to sending===<br />
<br />
=== Sensing ===<br />
<br />
Diffused HSL molecules are identified via [https://2014.igem.org/Team:ETH_Zurich/expresults#Quorum_Sensing quorum sensing machinery]. The [https://2014.igem.org/Team:ETH_Zurich/modeling/qs transfer function] between concentration of sensed HSL to promoter activation deviates from the ideal one due to leakiness and crosstalk between HSL molecules, regulatory proteins and promoters (we characterized it for [http://parts.igem.org/Part:BBa_R0062 Lux promoter (BBa_R0062)], [http://parts.igem.org/Part:BBa_R0079 Las promoter (BBa_R0079)] and [http://parts.igem.org/Part:BBa_R0071 Rhl promoter (BBa_R0071)]). To limit the risk of error propagation, we implemented a [http://parts.igem.org/Part:BBa_K1541000 riboregulated system].<br />
<br />
=== Computing ===<br />
<br />
[https://2014.igem.org/Team:ETH_Zurich/expresults#Integrases Integrases] compute an [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR logic gate] via single switching or double switching of a terminator. Their behavior depends on a set of [https://2014.igem.org/Team:ETH_Zurich/modeling/int parameters].<br />
<br />
=== Producing ===<br />
<br />
To propagate the computation results, the output has to be translated again into a quorum sensing molecule. Therefore a synthase is expressed to produce a new AHL signal.<br />
<br />
=== Memorising ===<br />
<br />
Alongside the AHL synthase, GFP is produced according to the XOR output. Th fluorescent protein acts both as long term memory for the colony state and as visual signal, storing and showing the progressive pattern formation.<br />
<br />
=== Sending ===<br />
<br />
The produced AHL diffuses through the channels to the rows below, becoming the input of the following layer. The speed of this directed diffusion determines the time needed from the pattern to emerge. You can find out more information about diffusion and chip design on our [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel modeling page], our [https://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion_On_Chip experimental results page] and our [https://2014.igem.org/Team:ETH_Zurich/lab/chip chip page].<br />
<br />
<br />
<br />
<br />
<br/><br />
<br/><br />
<br/><br />
[[File:ETH_Zurich_Information_Processing_chip.png|center|600px|thumb|'''Figure 2'''The information pathway in the project Mosai''coli'' on chip level. After initializing the signal, it propagates through the wells with directed diffusion. In each well, bacterial colonies have to be able to proceed in the information pathway: detecting (sensing), integrating (computing), producing and sending. These successive iterations leads to possible error propagation. Robustness is one major issue of our system.]]<br />
<br />
<html></article></html><br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/project/infoproTeam:ETH Zurich/project/infopro2014-10-18T02:05:43Z<p>Mbahls: /* Why we chose this track */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Information Processing}}<br />
<html><article></html><br />
<br />
== Information Processing ==<br />
<br />
<br />
=== About the track ===<br />
<br />
<br />
[https://2014.igem.org/Tracks/Information_Processing ''«Information Processing in iGEM covers a diverse range of projects. IP teams are not trying to solve a real world problem with practical applications, but to tackle an interesting problem that might otherwise not attract attention. Teams enter this track if they are attempting projects such as building elements of a biological computer, creating a game using biology or working on a signal processing challenges. Engineering ways to make biological systems perform computation is one of the core goals of synthetic biology. We generally work at the DNA level, engineering systems to function using BioBricks. In most biological systems, protein-protein interactions are where the majority of processing takes place. Being able to design proteins to accomplish computation would allow for systems to function on a much faster timescale than the current transcription-translation paradigm»'' (iGEM mainpage)]<br />
<br />
=== Why we chose this track ===<br />
<br />
With our project, Mosai''coli'', we investigate the emergence of complex patterns from simple mathematical rules. Such rules can be reduced to Boolean logic gates, in our case XOR gates. The computations were implemented with integrases, proteins modifing DNA between specific sites. These modifications in turn influence the expression of other proteins which can then indicate the previous change on the genetic level and chemically wire the information to the next cells, again on the protein level. This wiring of information and repeated information processing allows the construction of cellular automatas and eventually biocomputers.<br />
<br />
=== The goal: Pattern emergence via information processing===<br />
We implement a cellular automaton in bacterial colonies. Each bacterial colony is a core, computing an XOR gate. A sensor device detects the inputs, HSL molecules. Then, the cell integrates the signal through a logic gate, performed by proteins, the serine integrases. A necessary post processing step generates the production of HSL molecules. Meanwhile, GFP, a visual read out, longlastingly indicates the state of the well. The produced signal then propagates in a directive fashion through a millifluidic chip. This iterative process faces the challenges of leakiness, cross-talk, protein-level computation and diffusion.<br />
<br/><br />
<br/><br />
<br />
[[File:ETH_Zurich2014_info_processing1.png|center|800px|thumb|'''Figure 1'''The information pathway in the project Mosai''coli'' on a colony level.]]<br />
<br />
<br />
=== The steps involved: From sensing to sending===<br />
<br />
=== Sensing ===<br />
<br />
Diffused HSL molecules are identified via [https://2014.igem.org/Team:ETH_Zurich/expresults#Quorum_Sensing quorum sensing machinery]. The [https://2014.igem.org/Team:ETH_Zurich/modeling/qs transfer function] between concentration of sensed HSL to promoter activation deviates from the ideal one due to leakiness and crosstalk between HSL molecules, regulatory proteins and promoters (we characterized it for [http://parts.igem.org/Part:BBa_R0062 Lux promoter (BBa_R0062)], [http://parts.igem.org/Part:BBa_R0079 Las promoter (BBa_R0079)] and [http://parts.igem.org/Part:BBa_R0071 Rhl promoter (BBa_R0071)]). To limit the risk of error propagation, we implemented a [http://parts.igem.org/Part:BBa_K1541000 riboregulated system].<br />
<br />
=== Computing ===<br />
<br />
[https://2014.igem.org/Team:ETH_Zurich/expresults#Integrases Integrases] compute an [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR logic gate] via single switching or double switching of a terminator. Their behavior depends on a set of [https://2014.igem.org/Team:ETH_Zurich/modeling/int parameters].<br />
<br />
=== Producing ===<br />
<br />
To propagate the computation results, the output has to be translated again into a quorum sensing molecule. Therefore a synthase is expressed to produce a new AHL signal.<br />
<br />
=== Memorising ===<br />
<br />
Alongside the AHL synthase, GFP is produced according to the XOR output. Th fluorescent protein acts both as long term memory for the colony state and as visual signal, storing and showing the progressive pattern formation.<br />
<br />
=== Sending ===<br />
<br />
The produced AHL diffuses through the channels to the rows below, becoming the input of the following layer. The speed of this directed diffusion determines the time needed from the pattern to emerge. You can find out more information about diffusion and chip design on our [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel modeling page], our [https://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion_On_Chip experimental results page] and our [https://2014.igem.org/Team:ETH_Zurich/lab/chip chip page].<br />
<br />
<br />
<br />
<br />
<br/><br />
<br/><br />
<br/><br />
[[File:ETH_Zurich_Information_Processing_chip.png|center|600px|thumb|'''Figure 2'''The information pathway in the project Mosai''coli'' on chip level. After initializing the signal, it propagates through the wells with directed diffusion. In each well, bacterial colonies have to be able to proceed in the information pathway: detecting (sensing), integrating (computing), producing and sending. These successive iterations leads to possible error propagation. Robustness is one major issue of our system.]]<br />
<br />
<html></article></html><br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/project/infoproTeam:ETH Zurich/project/infopro2014-10-18T02:05:20Z<p>Mbahls: /* Why we chose this track */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Information Processing}}<br />
<html><article></html><br />
<br />
== Information Processing ==<br />
<br />
<br />
=== About the track ===<br />
<br />
<br />
[https://2014.igem.org/Tracks/Information_Processing ''«Information Processing in iGEM covers a diverse range of projects. IP teams are not trying to solve a real world problem with practical applications, but to tackle an interesting problem that might otherwise not attract attention. Teams enter this track if they are attempting projects such as building elements of a biological computer, creating a game using biology or working on a signal processing challenges. Engineering ways to make biological systems perform computation is one of the core goals of synthetic biology. We generally work at the DNA level, engineering systems to function using BioBricks. In most biological systems, protein-protein interactions are where the majority of processing takes place. Being able to design proteins to accomplish computation would allow for systems to function on a much faster timescale than the current transcription-translation paradigm»'' (iGEM mainpage)]<br />
<br />
=== Why we chose this track ===<br />
<br />
With our project, Mosai''coli'', we investigate the emergence of complex patterns from simple mathematical rules. This rules can be reduced to Boolean logic gates, in our case XOR gates. The computations were implemented with integrases, proteins modifing DNA between specific sites. These modifications in turn influence the expression of other proteins which can then indicate the previous change on the genetic level and chemically wire the information to the next cells, again on the protein level. This wiring of information and repeated information processing allows the construction of cellular automatas and eventually biocomputers.<br />
<br />
=== The goal: Pattern emergence via information processing===<br />
We implement a cellular automaton in bacterial colonies. Each bacterial colony is a core, computing an XOR gate. A sensor device detects the inputs, HSL molecules. Then, the cell integrates the signal through a logic gate, performed by proteins, the serine integrases. A necessary post processing step generates the production of HSL molecules. Meanwhile, GFP, a visual read out, longlastingly indicates the state of the well. The produced signal then propagates in a directive fashion through a millifluidic chip. This iterative process faces the challenges of leakiness, cross-talk, protein-level computation and diffusion.<br />
<br/><br />
<br/><br />
<br />
[[File:ETH_Zurich2014_info_processing1.png|center|800px|thumb|'''Figure 1'''The information pathway in the project Mosai''coli'' on a colony level.]]<br />
<br />
<br />
=== The steps involved: From sensing to sending===<br />
<br />
=== Sensing ===<br />
<br />
Diffused HSL molecules are identified via [https://2014.igem.org/Team:ETH_Zurich/expresults#Quorum_Sensing quorum sensing machinery]. The [https://2014.igem.org/Team:ETH_Zurich/modeling/qs transfer function] between concentration of sensed HSL to promoter activation deviates from the ideal one due to leakiness and crosstalk between HSL molecules, regulatory proteins and promoters (we characterized it for [http://parts.igem.org/Part:BBa_R0062 Lux promoter (BBa_R0062)], [http://parts.igem.org/Part:BBa_R0079 Las promoter (BBa_R0079)] and [http://parts.igem.org/Part:BBa_R0071 Rhl promoter (BBa_R0071)]). To limit the risk of error propagation, we implemented a [http://parts.igem.org/Part:BBa_K1541000 riboregulated system].<br />
<br />
=== Computing ===<br />
<br />
[https://2014.igem.org/Team:ETH_Zurich/expresults#Integrases Integrases] compute an [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR logic gate] via single switching or double switching of a terminator. Their behavior depends on a set of [https://2014.igem.org/Team:ETH_Zurich/modeling/int parameters].<br />
<br />
=== Producing ===<br />
<br />
To propagate the computation results, the output has to be translated again into a quorum sensing molecule. Therefore a synthase is expressed to produce a new AHL signal.<br />
<br />
=== Memorising ===<br />
<br />
Alongside the AHL synthase, GFP is produced according to the XOR output. Th fluorescent protein acts both as long term memory for the colony state and as visual signal, storing and showing the progressive pattern formation.<br />
<br />
=== Sending ===<br />
<br />
The produced AHL diffuses through the channels to the rows below, becoming the input of the following layer. The speed of this directed diffusion determines the time needed from the pattern to emerge. You can find out more information about diffusion and chip design on our [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel modeling page], our [https://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion_On_Chip experimental results page] and our [https://2014.igem.org/Team:ETH_Zurich/lab/chip chip page].<br />
<br />
<br />
<br />
<br />
<br/><br />
<br/><br />
<br/><br />
[[File:ETH_Zurich_Information_Processing_chip.png|center|600px|thumb|'''Figure 2'''The information pathway in the project Mosai''coli'' on chip level. After initializing the signal, it propagates through the wells with directed diffusion. In each well, bacterial colonies have to be able to proceed in the information pathway: detecting (sensing), integrating (computing), producing and sending. These successive iterations leads to possible error propagation. Robustness is one major issue of our system.]]<br />
<br />
<html></article></html><br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/project/infoproTeam:ETH Zurich/project/infopro2014-10-18T01:51:38Z<p>Mbahls: /* Information Processing */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Information Processing}}<br />
<html><article></html><br />
<br />
== Information Processing ==<br />
<br />
<br />
=== About the track ===<br />
<br />
<br />
[https://2014.igem.org/Tracks/Information_Processing ''«Information Processing in iGEM covers a diverse range of projects. IP teams are not trying to solve a real world problem with practical applications, but to tackle an interesting problem that might otherwise not attract attention. Teams enter this track if they are attempting projects such as building elements of a biological computer, creating a game using biology or working on a signal processing challenges. Engineering ways to make biological systems perform computation is one of the core goals of synthetic biology. We generally work at the DNA level, engineering systems to function using BioBricks. In most biological systems, protein-protein interactions are where the majority of processing takes place. Being able to design proteins to accomplish computation would allow for systems to function on a much faster timescale than the current transcription-translation paradigm»'' (iGEM mainpage)]<br />
<br />
=== Why we chose this track ===<br />
<br />
<br />
<br />
<br />
=== The goal: Pattern emergence via information processing===<br />
We implement a cellular automaton in bacterial colonies. Each bacterial colony is a core, computing an XOR gate. A sensor device detects the inputs, HSL molecules. Then, the cell integrates the signal through a logic gate, performed by proteins, the serine integrases. A necessary post processing step generates the production of HSL molecules. Meanwhile, GFP, a visual read out, longlastingly indicates the state of the well. The produced signal then propagates in a directive fashion through a millifluidic chip. This iterative process faces the challenges of leakiness, cross-talk, protein-level computation and diffusion.<br />
<br/><br />
<br/><br />
<br />
[[File:ETH_Zurich2014_info_processing1.png|center|800px|thumb|'''Figure 1'''The information pathway in the project Mosai''coli'' on a colony level.]]<br />
<br />
<br />
=== The steps involved: From sensing to sending===<br />
<br />
=== Sensing ===<br />
<br />
Diffused HSL molecules are identified via [https://2014.igem.org/Team:ETH_Zurich/expresults#Quorum_Sensing quorum sensing machinery]. The [https://2014.igem.org/Team:ETH_Zurich/modeling/qs transfer function] between concentration of sensed HSL to promoter activation deviates from the ideal one due to leakiness and crosstalk between HSL molecules, regulatory proteins and promoters (we characterized it for [http://parts.igem.org/Part:BBa_R0062 Lux promoter (BBa_R0062)], [http://parts.igem.org/Part:BBa_R0079 Las promoter (BBa_R0079)] and [http://parts.igem.org/Part:BBa_R0071 Rhl promoter (BBa_R0071)]). To limit the risk of error propagation, we implemented a [http://parts.igem.org/Part:BBa_K1541000 riboregulated system].<br />
<br />
=== Computing ===<br />
<br />
[https://2014.igem.org/Team:ETH_Zurich/expresults#Integrases Integrases] compute an [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR logic gate] via single switching or double switching of a terminator. Their behavior depends on a set of [https://2014.igem.org/Team:ETH_Zurich/modeling/int parameters].<br />
<br />
=== Producing ===<br />
<br />
To propagate the computation results, the output has to be translated again into a quorum sensing molecule. Therefore a synthase is expressed to produce a new AHL signal.<br />
<br />
=== Memorising ===<br />
<br />
Alongside the AHL synthase, GFP is produced according to the XOR output. Th fluorescent protein acts both as long term memory for the colony state and as visual signal, storing and showing the progressive pattern formation.<br />
<br />
=== Sending ===<br />
<br />
The produced AHL diffuses through the channels to the rows below, becoming the input of the following layer. The speed of this directed diffusion determines the time needed from the pattern to emerge. You can find out more information about diffusion and chip design on our [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel modeling page], our [https://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion_On_Chip experimental results page] and our [https://2014.igem.org/Team:ETH_Zurich/lab/chip chip page].<br />
<br />
<br />
<br />
<br />
<br/><br />
<br/><br />
<br/><br />
[[File:ETH_Zurich_Information_Processing_chip.png|center|600px|thumb|'''Figure 2'''The information pathway in the project Mosai''coli'' on chip level. After initializing the signal, it propagates through the wells with directed diffusion. In each well, bacterial colonies have to be able to proceed in the information pathway: detecting (sensing), integrating (computing), producing and sending. These successive iterations leads to possible error propagation. Robustness is one major issue of our system.]]<br />
<br />
<html></article></html><br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/project/infoproTeam:ETH Zurich/project/infopro2014-10-18T01:50:00Z<p>Mbahls: /* The goal: Pattern emergence */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Information Processing}}<br />
<html><article></html><br />
<br />
== Information Processing ==<br />
<br />
<br />
=== About the track ===<br />
<br />
<br />
[https://2014.igem.org/Tracks/Information_Processing ''«Information Processing in iGEM covers a diverse range of projects. IP teams are not trying to solve a real world problem with practical applications, but to tackle an interesting problem that might otherwise not attract attention. Teams enter this track if they are attempting projects such as building elements of a biological computer, creating a game using biology or working on a signal processing challenges. Engineering ways to make biological systems perform computation is one of the core goals of synthetic biology. We generally work at the DNA level, engineering systems to function using BioBricks. In most biological systems, protein-protein interactions are where the majority of processing takes place. Being able to design proteins to accomplish computation would allow for systems to function on a much faster timescale than the current transcription-translation paradigm»'' (iGEM mainpage)]<br />
<br />
=== Why we chose this track ===<br />
<br />
<br />
<br />
<br />
=== The goal: Pattern emergence via information processing===<br />
We implement a cellular automaton in bacterial colonies. Each bacterial colony is a core, computing an XOR gate. A sensor device detects the inputs, HSL molecules. Then, the cell integrates the signal through a logic gate, performed by proteins, the serine integrases. A necessary post processing step generates the production of HSL molecules. Meanwhile, GFP, a visual read out, longlastingly indicates the state of the well. The produced signal then propagates in a directive fashion through a millifluidic chip. This iterative process faces the challenges of leakiness, cross-talk, protein-level computation and diffusion.<br />
<br/><br />
<br/><br />
<br />
[[File:ETH_Zurich2014_info_processing1.png|center|800px|thumb|'''Figure 1'''The information pathway in the project Mosai''coli'' on a colony level.]]<br />
<br />
=== Sensing ===<br />
<br />
Diffused HSL molecules are identified via [https://2014.igem.org/Team:ETH_Zurich/expresults#Quorum_Sensing quorum sensing machinery]. The [https://2014.igem.org/Team:ETH_Zurich/modeling/qs transfer function] between concentration of sensed HSL to promoter activation deviates from the ideal one due to leakiness and crosstalk between HSL molecules, regulatory proteins and promoters (we characterized it for [http://parts.igem.org/Part:BBa_R0062 Lux promoter (BBa_R0062)], [http://parts.igem.org/Part:BBa_R0079 Las promoter (BBa_R0079)] and [http://parts.igem.org/Part:BBa_R0071 Rhl promoter (BBa_R0071)]). To limit the risk of error propagation, we implemented a [http://parts.igem.org/Part:BBa_K1541000 riboregulated system].<br />
<br />
=== Computing ===<br />
<br />
[https://2014.igem.org/Team:ETH_Zurich/expresults#Integrases Integrases] compute an [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR logic gate] via single switching or double switching of a terminator. Their behavior depends on a set of [https://2014.igem.org/Team:ETH_Zurich/modeling/int parameters].<br />
<br />
=== Producing ===<br />
<br />
To propagate the computation results, the output has to be translated again into a quorum sensing molecule. Therefore a synthase is expressed to produce a new AHL signal.<br />
<br />
=== Memorising ===<br />
<br />
Alongside the AHL synthase, GFP is produced according to the XOR output. Th fluorescent protein acts both as long term memory for the colony state and as visual signal, storing and showing the progressive pattern formation.<br />
<br />
=== Sending ===<br />
<br />
The produced AHL diffuses through the channels to the rows below, becoming the input of the following layer. The speed of this directed diffusion determines the time needed from the pattern to emerge. You can find out more information about diffusion and chip design on our [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel modeling page], our [https://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion_On_Chip experimental results page] and our [https://2014.igem.org/Team:ETH_Zurich/lab/chip chip page].<br />
<br />
<br />
<br />
<br />
<br/><br />
<br/><br />
<br/><br />
[[File:ETH_Zurich_Information_Processing_chip.png|center|600px|thumb|'''Figure 2'''The information pathway in the project Mosai''coli'' on chip level. After initializing the signal, it propagates through the wells with directed diffusion. In each well, bacterial colonies have to be able to proceed in the information pathway: detecting (sensing), integrating (computing), producing and sending. These successive iterations leads to possible error propagation. Robustness is one major issue of our system.]]<br />
<br />
<html></article></html><br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/project/infoproTeam:ETH Zurich/project/infopro2014-10-18T01:49:11Z<p>Mbahls: /* Pattern Emergence */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Information Processing}}<br />
<html><article></html><br />
<br />
== Information Processing ==<br />
<br />
<br />
=== About the track ===<br />
<br />
<br />
[https://2014.igem.org/Tracks/Information_Processing ''«Information Processing in iGEM covers a diverse range of projects. IP teams are not trying to solve a real world problem with practical applications, but to tackle an interesting problem that might otherwise not attract attention. Teams enter this track if they are attempting projects such as building elements of a biological computer, creating a game using biology or working on a signal processing challenges. Engineering ways to make biological systems perform computation is one of the core goals of synthetic biology. We generally work at the DNA level, engineering systems to function using BioBricks. In most biological systems, protein-protein interactions are where the majority of processing takes place. Being able to design proteins to accomplish computation would allow for systems to function on a much faster timescale than the current transcription-translation paradigm»'' (iGEM mainpage)]<br />
<br />
=== Why we chose this track ===<br />
<br />
<br />
<br />
<br />
=== The goal: Pattern emergence ===<br />
We implement a cellular automaton in bacterial colonies. Each bacterial colony is a core, computing an XOR gate. A sensor device detects the inputs, HSL molecules. Then, the cell integrates the signal through a logic gate, performed by proteins, the serine integrases. A necessary post processing step generates the production of HSL molecules. Meanwhile, GFP, a visual read out, longlastingly indicates the state of the well. The produced signal then propagates in a directive fashion through a millifluidic chip. This iterative process faces the challenges of leakiness, cross-talk, protein-level computation and diffusion.<br />
<br/><br />
<br/><br />
<br />
[[File:ETH_Zurich2014_info_processing1.png|center|800px|thumb|'''Figure 1'''The information pathway in the project Mosai''coli'' on a colony level.]]<br />
<br />
=== Sensing ===<br />
<br />
Diffused HSL molecules are identified via [https://2014.igem.org/Team:ETH_Zurich/expresults#Quorum_Sensing quorum sensing machinery]. The [https://2014.igem.org/Team:ETH_Zurich/modeling/qs transfer function] between concentration of sensed HSL to promoter activation deviates from the ideal one due to leakiness and crosstalk between HSL molecules, regulatory proteins and promoters (we characterized it for [http://parts.igem.org/Part:BBa_R0062 Lux promoter (BBa_R0062)], [http://parts.igem.org/Part:BBa_R0079 Las promoter (BBa_R0079)] and [http://parts.igem.org/Part:BBa_R0071 Rhl promoter (BBa_R0071)]). To limit the risk of error propagation, we implemented a [http://parts.igem.org/Part:BBa_K1541000 riboregulated system].<br />
<br />
=== Computing ===<br />
<br />
[https://2014.igem.org/Team:ETH_Zurich/expresults#Integrases Integrases] compute an [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR logic gate] via single switching or double switching of a terminator. Their behavior depends on a set of [https://2014.igem.org/Team:ETH_Zurich/modeling/int parameters].<br />
<br />
=== Producing ===<br />
<br />
To propagate the computation results, the output has to be translated again into a quorum sensing molecule. Therefore a synthase is expressed to produce a new AHL signal.<br />
<br />
=== Memorising ===<br />
<br />
Alongside the AHL synthase, GFP is produced according to the XOR output. Th fluorescent protein acts both as long term memory for the colony state and as visual signal, storing and showing the progressive pattern formation.<br />
<br />
=== Sending ===<br />
<br />
The produced AHL diffuses through the channels to the rows below, becoming the input of the following layer. The speed of this directed diffusion determines the time needed from the pattern to emerge. You can find out more information about diffusion and chip design on our [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel modeling page], our [https://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion_On_Chip experimental results page] and our [https://2014.igem.org/Team:ETH_Zurich/lab/chip chip page].<br />
<br />
<br />
<br />
<br />
<br/><br />
<br/><br />
<br/><br />
[[File:ETH_Zurich_Information_Processing_chip.png|center|600px|thumb|'''Figure 2'''The information pathway in the project Mosai''coli'' on chip level. After initializing the signal, it propagates through the wells with directed diffusion. In each well, bacterial colonies have to be able to proceed in the information pathway: detecting (sensing), integrating (computing), producing and sending. These successive iterations leads to possible error propagation. Robustness is one major issue of our system.]]<br />
<br />
<html></article></html><br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/project/infoproTeam:ETH Zurich/project/infopro2014-10-18T01:45:14Z<p>Mbahls: /* Track: Information Processing */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Information Processing}}<br />
<html><article></html><br />
<br />
== Information Processing ==<br />
<br />
<br />
=== About the track ===<br />
<br />
<br />
[https://2014.igem.org/Tracks/Information_Processing ''«Information Processing in iGEM covers a diverse range of projects. IP teams are not trying to solve a real world problem with practical applications, but to tackle an interesting problem that might otherwise not attract attention. Teams enter this track if they are attempting projects such as building elements of a biological computer, creating a game using biology or working on a signal processing challenges. Engineering ways to make biological systems perform computation is one of the core goals of synthetic biology. We generally work at the DNA level, engineering systems to function using BioBricks. In most biological systems, protein-protein interactions are where the majority of processing takes place. Being able to design proteins to accomplish computation would allow for systems to function on a much faster timescale than the current transcription-translation paradigm»'' (iGEM mainpage)]<br />
<br />
=== Pattern Emergence ===<br />
We implement a cellular automaton in bacterial colonies. Each bacterial colony is a core, computing an XOR gate. A sensor device detects the inputs, HSL molecules. Then, the cell integrates the signal through a logic gate, performed by proteins, the serine integrases. A necessary post processing step generates the production of HSL molecules. Meanwhile, GFP, a visual read out, longlastingly indicates the state of the well. The produced signal then propagates in a directive fashion through a millifluidic chip. This iterative process faces the challenges of leakiness, cross-talk, protein-level computation and diffusion.<br />
<br/><br />
<br/><br />
<br />
[[File:ETH_Zurich2014_info_processing1.png|center|800px|thumb|'''Figure 1'''The information pathway in the project Mosai''coli'' on a colony level.]]<br />
<br />
=== Sensing ===<br />
<br />
Diffused HSL molecules are identified via [https://2014.igem.org/Team:ETH_Zurich/expresults#Quorum_Sensing quorum sensing machinery]. The [https://2014.igem.org/Team:ETH_Zurich/modeling/qs transfer function] between concentration of sensed HSL to promoter activation deviates from the ideal one due to leakiness and crosstalk between HSL molecules, regulatory proteins and promoters (we characterized it for [http://parts.igem.org/Part:BBa_R0062 Lux promoter (BBa_R0062)], [http://parts.igem.org/Part:BBa_R0079 Las promoter (BBa_R0079)] and [http://parts.igem.org/Part:BBa_R0071 Rhl promoter (BBa_R0071)]). To limit the risk of error propagation, we implemented a [http://parts.igem.org/Part:BBa_K1541000 riboregulated system].<br />
<br />
=== Computing ===<br />
<br />
[https://2014.igem.org/Team:ETH_Zurich/expresults#Integrases Integrases] compute an [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR logic gate] via single switching or double switching of a terminator. Their behavior depends on a set of [https://2014.igem.org/Team:ETH_Zurich/modeling/int parameters].<br />
<br />
=== Producing ===<br />
<br />
To propagate the computation results, the output has to be translated again into a quorum sensing molecule. Therefore a synthase is expressed to produce a new AHL signal.<br />
<br />
=== Memorising ===<br />
<br />
Alongside the AHL synthase, GFP is produced according to the XOR output. Th fluorescent protein acts both as long term memory for the colony state and as visual signal, storing and showing the progressive pattern formation.<br />
<br />
=== Sending ===<br />
<br />
The produced AHL diffuses through the channels to the rows below, becoming the input of the following layer. The speed of this directed diffusion determines the time needed from the pattern to emerge. You can find out more information about diffusion and chip design on our [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel modeling page], our [https://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion_On_Chip experimental results page] and our [https://2014.igem.org/Team:ETH_Zurich/lab/chip chip page].<br />
<br />
<br />
<br />
<br />
<br/><br />
<br/><br />
<br/><br />
[[File:ETH_Zurich_Information_Processing_chip.png|center|600px|thumb|'''Figure 2'''The information pathway in the project Mosai''coli'' on chip level. After initializing the signal, it propagates through the wells with directed diffusion. In each well, bacterial colonies have to be able to proceed in the information pathway: detecting (sensing), integrating (computing), producing and sending. These successive iterations leads to possible error propagation. Robustness is one major issue of our system.]]<br />
<br />
<html></article></html><br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/project/infoproTeam:ETH Zurich/project/infopro2014-10-18T01:44:51Z<p>Mbahls: /* About the track */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Information Processing}}<br />
<html><article></html><br />
<br />
== Track: Information Processing ==<br />
<br />
<br />
=== About the track ===<br />
<br />
<br />
[https://2014.igem.org/Tracks/Information_Processing ''«Information Processing in iGEM covers a diverse range of projects. IP teams are not trying to solve a real world problem with practical applications, but to tackle an interesting problem that might otherwise not attract attention. Teams enter this track if they are attempting projects such as building elements of a biological computer, creating a game using biology or working on a signal processing challenges. Engineering ways to make biological systems perform computation is one of the core goals of synthetic biology. We generally work at the DNA level, engineering systems to function using BioBricks. In most biological systems, protein-protein interactions are where the majority of processing takes place. Being able to design proteins to accomplish computation would allow for systems to function on a much faster timescale than the current transcription-translation paradigm»'' (iGEM mainpage)]<br />
<br />
=== Pattern Emergence ===<br />
We implement a cellular automaton in bacterial colonies. Each bacterial colony is a core, computing an XOR gate. A sensor device detects the inputs, HSL molecules. Then, the cell integrates the signal through a logic gate, performed by proteins, the serine integrases. A necessary post processing step generates the production of HSL molecules. Meanwhile, GFP, a visual read out, longlastingly indicates the state of the well. The produced signal then propagates in a directive fashion through a millifluidic chip. This iterative process faces the challenges of leakiness, cross-talk, protein-level computation and diffusion.<br />
<br/><br />
<br/><br />
<br />
[[File:ETH_Zurich2014_info_processing1.png|center|800px|thumb|'''Figure 1'''The information pathway in the project Mosai''coli'' on a colony level.]]<br />
<br />
=== Sensing ===<br />
<br />
Diffused HSL molecules are identified via [https://2014.igem.org/Team:ETH_Zurich/expresults#Quorum_Sensing quorum sensing machinery]. The [https://2014.igem.org/Team:ETH_Zurich/modeling/qs transfer function] between concentration of sensed HSL to promoter activation deviates from the ideal one due to leakiness and crosstalk between HSL molecules, regulatory proteins and promoters (we characterized it for [http://parts.igem.org/Part:BBa_R0062 Lux promoter (BBa_R0062)], [http://parts.igem.org/Part:BBa_R0079 Las promoter (BBa_R0079)] and [http://parts.igem.org/Part:BBa_R0071 Rhl promoter (BBa_R0071)]). To limit the risk of error propagation, we implemented a [http://parts.igem.org/Part:BBa_K1541000 riboregulated system].<br />
<br />
=== Computing ===<br />
<br />
[https://2014.igem.org/Team:ETH_Zurich/expresults#Integrases Integrases] compute an [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR logic gate] via single switching or double switching of a terminator. Their behavior depends on a set of [https://2014.igem.org/Team:ETH_Zurich/modeling/int parameters].<br />
<br />
=== Producing ===<br />
<br />
To propagate the computation results, the output has to be translated again into a quorum sensing molecule. Therefore a synthase is expressed to produce a new AHL signal.<br />
<br />
=== Memorising ===<br />
<br />
Alongside the AHL synthase, GFP is produced according to the XOR output. Th fluorescent protein acts both as long term memory for the colony state and as visual signal, storing and showing the progressive pattern formation.<br />
<br />
=== Sending ===<br />
<br />
The produced AHL diffuses through the channels to the rows below, becoming the input of the following layer. The speed of this directed diffusion determines the time needed from the pattern to emerge. You can find out more information about diffusion and chip design on our [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel modeling page], our [https://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion_On_Chip experimental results page] and our [https://2014.igem.org/Team:ETH_Zurich/lab/chip chip page].<br />
<br />
<br />
<br />
<br />
<br/><br />
<br/><br />
<br/><br />
[[File:ETH_Zurich_Information_Processing_chip.png|center|600px|thumb|'''Figure 2'''The information pathway in the project Mosai''coli'' on chip level. After initializing the signal, it propagates through the wells with directed diffusion. In each well, bacterial colonies have to be able to proceed in the information pathway: detecting (sensing), integrating (computing), producing and sending. These successive iterations leads to possible error propagation. Robustness is one major issue of our system.]]<br />
<br />
<html></article></html><br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/project/infoproTeam:ETH Zurich/project/infopro2014-10-18T01:44:35Z<p>Mbahls: /* About the track */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Information Processing}}<br />
<html><article></html><br />
<br />
== Track: Information Processing ==<br />
<br />
<br />
=== About the track ===<br />
<br />
<br />
[https://2014.igem.org/Tracks/Information_Processing ''«Information Processing in iGEM covers a diverse range of projects. IP teams are not trying to solve a real world problem with practical applications, but to tackle an interesting problem that might otherwise not attract attention. Teams enter this track if they are attempting projects such as building elements of a biological computer, creating a game using biology or working on a signal processing challenges. Engineering ways to make biological systems perform computation is one of the core goals of synthetic biology. We generally work at the DNA level, engineering systems to function using BioBricks. In most biological systems, protein-protein interactions are where the majority of processing takes place. Being able to design proteins to accomplish computation would allow for systems to function on a much faster timescale than the current transcription-translation paradigm»''(iGEM mainpage)]<br />
<br />
=== Pattern Emergence ===<br />
We implement a cellular automaton in bacterial colonies. Each bacterial colony is a core, computing an XOR gate. A sensor device detects the inputs, HSL molecules. Then, the cell integrates the signal through a logic gate, performed by proteins, the serine integrases. A necessary post processing step generates the production of HSL molecules. Meanwhile, GFP, a visual read out, longlastingly indicates the state of the well. The produced signal then propagates in a directive fashion through a millifluidic chip. This iterative process faces the challenges of leakiness, cross-talk, protein-level computation and diffusion.<br />
<br/><br />
<br/><br />
<br />
[[File:ETH_Zurich2014_info_processing1.png|center|800px|thumb|'''Figure 1'''The information pathway in the project Mosai''coli'' on a colony level.]]<br />
<br />
=== Sensing ===<br />
<br />
Diffused HSL molecules are identified via [https://2014.igem.org/Team:ETH_Zurich/expresults#Quorum_Sensing quorum sensing machinery]. The [https://2014.igem.org/Team:ETH_Zurich/modeling/qs transfer function] between concentration of sensed HSL to promoter activation deviates from the ideal one due to leakiness and crosstalk between HSL molecules, regulatory proteins and promoters (we characterized it for [http://parts.igem.org/Part:BBa_R0062 Lux promoter (BBa_R0062)], [http://parts.igem.org/Part:BBa_R0079 Las promoter (BBa_R0079)] and [http://parts.igem.org/Part:BBa_R0071 Rhl promoter (BBa_R0071)]). To limit the risk of error propagation, we implemented a [http://parts.igem.org/Part:BBa_K1541000 riboregulated system].<br />
<br />
=== Computing ===<br />
<br />
[https://2014.igem.org/Team:ETH_Zurich/expresults#Integrases Integrases] compute an [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR logic gate] via single switching or double switching of a terminator. Their behavior depends on a set of [https://2014.igem.org/Team:ETH_Zurich/modeling/int parameters].<br />
<br />
=== Producing ===<br />
<br />
To propagate the computation results, the output has to be translated again into a quorum sensing molecule. Therefore a synthase is expressed to produce a new AHL signal.<br />
<br />
=== Memorising ===<br />
<br />
Alongside the AHL synthase, GFP is produced according to the XOR output. Th fluorescent protein acts both as long term memory for the colony state and as visual signal, storing and showing the progressive pattern formation.<br />
<br />
=== Sending ===<br />
<br />
The produced AHL diffuses through the channels to the rows below, becoming the input of the following layer. The speed of this directed diffusion determines the time needed from the pattern to emerge. You can find out more information about diffusion and chip design on our [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel modeling page], our [https://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion_On_Chip experimental results page] and our [https://2014.igem.org/Team:ETH_Zurich/lab/chip chip page].<br />
<br />
<br />
<br />
<br />
<br/><br />
<br/><br />
<br/><br />
[[File:ETH_Zurich_Information_Processing_chip.png|center|600px|thumb|'''Figure 2'''The information pathway in the project Mosai''coli'' on chip level. After initializing the signal, it propagates through the wells with directed diffusion. In each well, bacterial colonies have to be able to proceed in the information pathway: detecting (sensing), integrating (computing), producing and sending. These successive iterations leads to possible error propagation. Robustness is one major issue of our system.]]<br />
<br />
<html></article></html><br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/project/infoproTeam:ETH Zurich/project/infopro2014-10-18T01:42:43Z<p>Mbahls: /* About the track */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Information Processing}}<br />
<html><article></html><br />
<br />
== Track: Information Processing ==<br />
<br />
<br />
=== About the track ===<br />
<br />
''«Information Processing in iGEM covers a diverse range of projects. IP teams are not trying to solve a real world problem with practical applications, but to tackle an interesting problem that might otherwise not attract attention. Teams enter this track if they are attempting projects such as building elements of a biological computer, creating a game using biology or working on a signal processing challenges. Engineering ways to make biological systems perform computation is one of the core goals of synthetic biology. We generally work at the DNA level, engineering systems to function using BioBricks. In most biological systems, protein-protein interactions are where the majority of processing takes place. Being able to design proteins to accomplish computation would allow for systems to function on a much faster timescale than the current transcription-translation paradigm..»'' <br />
<br />
[https://2014.igem.org/Tracks/Information_Processing (iGEM)] </center><br />
<br/><br />
<br/><br />
<br />
=== Pattern Emergence ===<br />
We implement a cellular automaton in bacterial colonies. Each bacterial colony is a core, computing an XOR gate. A sensor device detects the inputs, HSL molecules. Then, the cell integrates the signal through a logic gate, performed by proteins, the serine integrases. A necessary post processing step generates the production of HSL molecules. Meanwhile, GFP, a visual read out, longlastingly indicates the state of the well. The produced signal then propagates in a directive fashion through a millifluidic chip. This iterative process faces the challenges of leakiness, cross-talk, protein-level computation and diffusion.<br />
<br/><br />
<br/><br />
<br />
[[File:ETH_Zurich2014_info_processing1.png|center|800px|thumb|'''Figure 1'''The information pathway in the project Mosai''coli'' on a colony level.]]<br />
<br />
=== Sensing ===<br />
<br />
Diffused HSL molecules are identified via [https://2014.igem.org/Team:ETH_Zurich/expresults#Quorum_Sensing quorum sensing machinery]. The [https://2014.igem.org/Team:ETH_Zurich/modeling/qs transfer function] between concentration of sensed HSL to promoter activation deviates from the ideal one due to leakiness and crosstalk between HSL molecules, regulatory proteins and promoters (we characterized it for [http://parts.igem.org/Part:BBa_R0062 Lux promoter (BBa_R0062)], [http://parts.igem.org/Part:BBa_R0079 Las promoter (BBa_R0079)] and [http://parts.igem.org/Part:BBa_R0071 Rhl promoter (BBa_R0071)]). To limit the risk of error propagation, we implemented a [http://parts.igem.org/Part:BBa_K1541000 riboregulated system].<br />
<br />
=== Computing ===<br />
<br />
[https://2014.igem.org/Team:ETH_Zurich/expresults#Integrases Integrases] compute an [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR logic gate] via single switching or double switching of a terminator. Their behavior depends on a set of [https://2014.igem.org/Team:ETH_Zurich/modeling/int parameters].<br />
<br />
=== Producing ===<br />
<br />
To propagate the computation results, the output has to be translated again into a quorum sensing molecule. Therefore a synthase is expressed to produce a new AHL signal.<br />
<br />
=== Memorising ===<br />
<br />
Alongside the AHL synthase, GFP is produced according to the XOR output. Th fluorescent protein acts both as long term memory for the colony state and as visual signal, storing and showing the progressive pattern formation.<br />
<br />
=== Sending ===<br />
<br />
The produced AHL diffuses through the channels to the rows below, becoming the input of the following layer. The speed of this directed diffusion determines the time needed from the pattern to emerge. You can find out more information about diffusion and chip design on our [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel modeling page], our [https://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion_On_Chip experimental results page] and our [https://2014.igem.org/Team:ETH_Zurich/lab/chip chip page].<br />
<br />
<br />
<br />
<br />
<br/><br />
<br/><br />
<br/><br />
[[File:ETH_Zurich_Information_Processing_chip.png|center|600px|thumb|'''Figure 2'''The information pathway in the project Mosai''coli'' on chip level. After initializing the signal, it propagates through the wells with directed diffusion. In each well, bacterial colonies have to be able to proceed in the information pathway: detecting (sensing), integrating (computing), producing and sending. These successive iterations leads to possible error propagation. Robustness is one major issue of our system.]]<br />
<br />
<html></article></html><br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/expresults/integrasesTeam:ETH Zurich/expresults/integrases2014-10-18T01:19:31Z<p>Mbahls: /* Integrases */</p>
<hr />
<div>== Integrases ==<br />
<br />
The design of our [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR gates] was based on [https://2014.igem.org/Team:ETH_Zurich/modeling/xor#Biological_Principles integrase logic]<sup>[[Team:ETH_Zurich/project/references#refBonnet|[9]]]</sup>. This means, depending on the input molecules, integrases can be expressed, subsequently switch a terminator sequence previously blocking gene expression, and then the output gene can be transcribed. This approach is explained [https://2014.igem.org/Team:ETH_Zurich/modeling/xor#Biological_Principles here].<br />
<br />
<br />
In order to characterize the integrase system described above, we first combined the [http://parts.igem.org/Part:BBa_R0062 pLux promoter (BBa_R0062)] with one of our [https://2014.igem.org/Team:ETH_Zurich/modeling/int integrase] genes ''bxb1'', followed directly by a red fluorescent protein (RFP, mCherry) to make the expression accessible. Also, this system includes an XOR buffer gate per default blocking transcription of sfGFP. Upon BXB1 activity and switching the gate into ON-state, the terminator should have been removed and sfGFP should have been expressed. We intially designed three different constructs for characterization of the recombinases and their cross-activity. However, the measurement of fluorescent proteins, with both a [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#Tecan_Infinite_M200_Pro.E2.84.A2 plate reader] and a [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#BD_LSRFortessa.E2.84.A2_Flow_Cytometer_System flow cytometer], did not indicate sfGFP expression due to recombinase activity. Nevertheless, RFP was clearly detectable upon induction in plate reader experiments suggesting that the induction itself worked (see figure 5 and 6).<br />
<br />
<br />
{|class="wikitable resized" style="background-color: white; border: 0px+important;"<br />
|[[File:ETH Zurich 2014 250min Integrase mCherry corrected.png|center|600px|thumb|'''Figure 5''' '''Expression of red fluorescent protein (RFP) and green fluorescent protein (GFP) 250 min after induction with various concentrations of 3OC6-HSL (10<sup>-13</sup> M to 10<sup>-4</sup> M).''' RFP is under the control of the [http://parts.igem.org/Part:BBa_R0062 pLux promoter (BBa_R0062)] together with the integrase BXB1. Upon expression of BXB1 a buffer gate should have been swtiched to ON-state producing GFP. Data points are mean values of triplicate measurements in 96-well microtiter plates &plusmn; standard deviation. For the full data set and kinetics please [https://2014.igem.org/Team:ETH_Zurich/contact contact] us or visit the [https://2014.igem.org/Team:ETH_Zurich/data/raw raw data] page.]]<br />
|[[File:ETH Zurich 2014 610minIntegrase corrected.png|center|600px|thumb|'''Figure 6''' '''Expression of red fluorescent protein (RFP) and green fluorescent protein (GFP) 610 min after induction with various concentrations of 3OC6-HSL (10<sup>-13</sup> M to 10<sup>-4</sup> M).''' RFP is under the control of the [http://parts.igem.org/Part:BBa_R0062 pLux promoter (BBa_R0062)] together with the integrase BXB1. Upon expression of BXB1 a buffer gate should have been swtiched to ON-state producing GFP. Data points are mean values of triplicate measurements in 96-well microtiter plates &plusmn; standard deviation. For the full data set and kinetics please [https://2014.igem.org/Team:ETH_Zurich/contact contact] us or visit the [https://2014.igem.org/Team:ETH_Zurich/data/raw raw data] page.]]<br />
|}<br />
<br />
<br />
As our constructs did not show the expected functionality, we decided to directly use the plasmids described by Bonnet ''et al''.<sup>[[Team:ETH_Zurich/project/references#refBonnet|[9]]]</sup> which where obtained from addgene ([http://www.addgene.org/44456 Dual-recombinase-controller], [http://www.addgene.org/44453/ XOR gate-V2.0]). The data available from the original experiment by Bonnet<sup>[[Team:ETH_Zurich/project/references#refBonnet|[9]]]</sup> was used in our model to retrieve the [https://2014.igem.org/Team:ETH_Zurich/modeling/int missing parameters of integrases]. However, we were using a [http://www.openwetware.org/wiki/E._coli_genotypes#TOP10_.28Invitrogen.29 TOP10] strain not expressing TetR by default (as compared to [http://www.expressys.com/main_strains.html DH5alphaZ1]) and as a result our strain had to be co-transformed with an additional plasmid encoding TetR. Also, we used defined [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#Defined_M9_medium_with_0.4_.25_glycerol M9 medium] with 0.4% glycerol and 1% CAA instead of proprietary defined medium ([http://teknova-blog.com/hi-def-azure-media Teknova Hi-Def Azure medium]). As of today, this set-up did not allow us to get the integrase XOR gate running. The fluorescence readout for ON-states (exactly one input, either 0.1% L-arabinose or 200 ng/mL anhydrous tetracycline) and the OFF-states was not as expected (see figure 7). The OFF-state should not show increased fluorescence over time, while the ON-states should increase significantly after 4 h and continue to increase over the whole time span<sup>[[Team:ETH_Zurich/project/references#refBonnet|[9]]]</sup>. We are not giving up on this and are proceeding with debugging our construct further and hope to find a solution until the Giant Jamboree in Boston.<br />
<br />
<br />
{{:Team:ETH_Zurich/tpl/topbutton|green}}<br />
<br />
<br />
[[File:ETH Zurich 2014 integrases addgene plasmids corrected.png|center|600px|thumb|'''Figure 7 Expression of green fluorescent protein (GFP) after induction of [https://2014.igem.org/Team:ETH_Zurich/modeling/xor#Biological_Principles XOR integrase logic].''' Upon induction of integrase BXB1 (t=0 min) a [https://2014.igem.org/Team:ETH_Zurich/lab/sequences#BUFFER_Gate_Construct buffer gate] should have been switched to ON-state producing GFP only if either L-arabinose (L-ara) or anhydrous tetracycline (aTc) is present. Data points are mean values of triplicate measurements in 96-well microtiter plates &plusmn; standard deviation. For the full data set and kinetics please [https://2014.igem.org/Team:ETH_Zurich/contact contact] us or visit the [https://2014.igem.org/Team:ETH_Zurich/data/raw raw data] page.]]</div>Mbahlshttp://2014.igem.org/File:ETH_Zurich_2014_integrases_addgene_plasmids_corrected.pngFile:ETH Zurich 2014 integrases addgene plasmids corrected.png2014-10-18T01:18:59Z<p>Mbahls: </p>
<hr />
<div></div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/lab/chipTeam:ETH Zurich/lab/chip2014-10-18T01:10:50Z<p>Mbahls: /* Time-Lapse Movies */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Millifluidic Chip & Rapid Prototyping}}<br />
<br />
<html><article style='min-height:800px'></html><br />
==Overview==<br />
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).<br />
<br />
[[File:ETH Zurich Rule 6.PNG|300px|thumb|left|'''Figure 1''' '''Classical grid from cellular automata theory''' (ON state=back, OFF state=white).]]<br />
[[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).]]<br />
<br />
<br />
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.<br />
<br><br />
<br><br />
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.<br />
<br />
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<html><article></html><br />
<br />
==Mold Design and 3D Print Exchange==<br />
<br />
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>. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_mesh_2_20140826.jpg|200px]]<br />
|[[File:ETH Zurich 2014 final mold model 2.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion plate model.jpeg|200px]]<br />
|-<br />
|'''Figure 3-a''' The first design for a gel-comb, a mold for a millifluidic PDMS chip and a corresponding box for the mold.<br />
|'''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. <br />
|'''Figure 3-c''' A design for a 96-well plate with connected wells, which allows automated measurements in a plate reader.<br />
|}<br />
<br />
<br />
'''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.<br />
'''<br />
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==3D-Printing and Rapid Prototyping==<br />
<br />
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. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 MakerBot.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerWare.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerBot ABS.jpg|x200px]]<br />
|-<br />
|'''Figure 4-a''' The MakerBot Replicator (2nd generation) we used to print our molds.<br />
|'''Figure 4-b''' 'Screenshot' of the MakerWare software we used to print our molds.<br />
|'''Figure 4-c''' A roll of ABS filament used by the 3D-printer.<br />
|}<br />
<br />
<br />
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).<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_comb_and_box.jpg|200px]]<br />
|[[File:ETH Zurich 2014 small grid.JPG|200px]]<br />
|[[File:ETH Zurich diffusion plate.JPG|200px]]<br />
|[[File:ETH Zurich 2014 96 well all connected.jpeg|200px]]<br />
|-<br />
|'''Figure 5-a''' Printed gel-comb and box. The box was autoclaved at 121 ⁰C. <br />
|'''Figure 5-b''' Printed millifluid grid with interconnected wells (edge length of 3 mm).<br />
|'''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).<br />
|'''Figure 5-d''' Printed 96-well plate, all wells (edge length of 5 mm) are connected.<br />
|}<br />
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<html><article id='Preparation'></html><br />
<br />
==PDMS Chip Preparation==<br />
<br />
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].<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_first_mold_with_PDMS.jpg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion mold.JPG|200px]]<br />
|[[File:ETH Zurich 2014 final mold.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 final mold closeup.jpeg|200px]]<br />
|-<br />
|'''Figure 6-a''' The very first mold design. PDMS stuck between the wells while removing it. <br />
|'''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).<br />
|'''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). <br />
|'''Figure 6-d''' Close up of the final mold design. The separate layers are clearly visible ('additive' manufacturing).<br />
|}<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 broken chip.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 2 well diffusion chip upsidedown.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 PDMS diffusion chip final.jpeg|200px]]<br />
|[[File:ETH_Zurich_2014_final_chip_zoom.png|200px]]<br />
|-<br />
|'''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.<br />
|'''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).<br />
|'''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). <br />
|'''Figure 7-d''' Close up of the final PDMS chip. The channels are well defined and even small structures separated evenly from the mold.<br />
|}<br />
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<html><article id='movies'></html><br />
<br />
==Time-Lapse Movies==<br />
<br />
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.<br />
<br />
<br />
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. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:100%; max-width: 650px; margin: auto;"<br />
|{{: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>}}<br />
|{{: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>}}<br />
|-<br />
|'''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.<br />
|'''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.<br />
|-<br />
|{{: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>}}<br />
|{{: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>}}<br />
|-<br />
|'''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.<br />
|'''Video 4 Cell communication experiment with [ 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.<br />
|-<br />
|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>}}<br />
|-<br />
|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.<br />
|}<br />
<br />
<br />
<html></article></html><br />
<br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/lab/chipTeam:ETH Zurich/lab/chip2014-10-18T01:08:31Z<p>Mbahls: /* Time-Lapse Movies */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Millifluidic Chip & Rapid Prototyping}}<br />
<br />
<html><article style='min-height:800px'></html><br />
==Overview==<br />
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).<br />
<br />
[[File:ETH Zurich Rule 6.PNG|300px|thumb|left|'''Figure 1''' '''Classical grid from cellular automata theory''' (ON state=back, OFF state=white).]]<br />
[[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).]]<br />
<br />
<br />
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.<br />
<br><br />
<br><br />
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.<br />
<br />
<html></article></html><br />
<br />
<br />
<html><article></html><br />
<br />
==Mold Design and 3D Print Exchange==<br />
<br />
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>. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_mesh_2_20140826.jpg|200px]]<br />
|[[File:ETH Zurich 2014 final mold model 2.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion plate model.jpeg|200px]]<br />
|-<br />
|'''Figure 3-a''' The first design for a gel-comb, a mold for a millifluidic PDMS chip and a corresponding box for the mold.<br />
|'''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. <br />
|'''Figure 3-c''' A design for a 96-well plate with connected wells, which allows automated measurements in a plate reader.<br />
|}<br />
<br />
<br />
'''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.<br />
'''<br />
<html></article></html><br />
<br />
<html><article></html><br />
<br />
==3D-Printing and Rapid Prototyping==<br />
<br />
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. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 MakerBot.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerWare.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerBot ABS.jpg|x200px]]<br />
|-<br />
|'''Figure 4-a''' The MakerBot Replicator (2nd generation) we used to print our molds.<br />
|'''Figure 4-b''' 'Screenshot' of the MakerWare software we used to print our molds.<br />
|'''Figure 4-c''' A roll of ABS filament used by the 3D-printer.<br />
|}<br />
<br />
<br />
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).<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_comb_and_box.jpg|200px]]<br />
|[[File:ETH Zurich 2014 small grid.JPG|200px]]<br />
|[[File:ETH Zurich diffusion plate.JPG|200px]]<br />
|[[File:ETH Zurich 2014 96 well all connected.jpeg|200px]]<br />
|-<br />
|'''Figure 5-a''' Printed gel-comb and box. The box was autoclaved at 121 ⁰C. <br />
|'''Figure 5-b''' Printed millifluid grid with interconnected wells (edge length of 3 mm).<br />
|'''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).<br />
|'''Figure 5-d''' Printed 96-well plate, all wells (edge length of 5 mm) are connected.<br />
|}<br />
<br />
<html></article></html><br />
<br />
<html><article id='Preparation'></html><br />
<br />
==PDMS Chip Preparation==<br />
<br />
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].<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_first_mold_with_PDMS.jpg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion mold.JPG|200px]]<br />
|[[File:ETH Zurich 2014 final mold.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 final mold closeup.jpeg|200px]]<br />
|-<br />
|'''Figure 6-a''' The very first mold design. PDMS stuck between the wells while removing it. <br />
|'''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).<br />
|'''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). <br />
|'''Figure 6-d''' Close up of the final mold design. The separate layers are clearly visible ('additive' manufacturing).<br />
|}<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 broken chip.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 2 well diffusion chip upsidedown.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 PDMS diffusion chip final.jpeg|200px]]<br />
|[[File:ETH_Zurich_2014_final_chip_zoom.png|200px]]<br />
|-<br />
|'''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.<br />
|'''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).<br />
|'''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). <br />
|'''Figure 7-d''' Close up of the final PDMS chip. The channels are well defined and even small structures separated evenly from the mold.<br />
|}<br />
<html></article></html><br />
<br />
<html><article id='movies'></html><br />
<br />
==Time-Lapse Movies==<br />
<br />
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.<br />
<br />
<br />
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. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:100%; max-width: 650px; margin: auto;"<br />
|{{: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>}}<br />
|{{: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>}}<br />
|-<br />
|'''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.<br />
|'''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.<br />
|-<br />
|{{: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>}}<br />
|{{: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>}}<br />
|-<br />
|'''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, 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). ~1850x faster than real-time.<br />
|'''Video 4 Cell communication experiment with [ 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). ~3450x faster than real-time.<br />
|-<br />
|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>}}<br />
|-<br />
|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.<br />
|}<br />
<br />
<br />
<html></article></html><br />
<br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/lab/chipTeam:ETH Zurich/lab/chip2014-10-18T01:06:29Z<p>Mbahls: </p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Millifluidic Chip & Rapid Prototyping}}<br />
<br />
<html><article style='min-height:800px'></html><br />
==Overview==<br />
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).<br />
<br />
[[File:ETH Zurich Rule 6.PNG|300px|thumb|left|'''Figure 1''' '''Classical grid from cellular automata theory''' (ON state=back, OFF state=white).]]<br />
[[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).]]<br />
<br />
<br />
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.<br />
<br><br />
<br><br />
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.<br />
<br />
<html></article></html><br />
<br />
<br />
<html><article></html><br />
<br />
==Mold Design and 3D Print Exchange==<br />
<br />
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>. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_mesh_2_20140826.jpg|200px]]<br />
|[[File:ETH Zurich 2014 final mold model 2.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion plate model.jpeg|200px]]<br />
|-<br />
|'''Figure 3-a''' The first design for a gel-comb, a mold for a millifluidic PDMS chip and a corresponding box for the mold.<br />
|'''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. <br />
|'''Figure 3-c''' A design for a 96-well plate with connected wells, which allows automated measurements in a plate reader.<br />
|}<br />
<br />
<br />
'''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.<br />
'''<br />
<html></article></html><br />
<br />
<html><article></html><br />
<br />
==3D-Printing and Rapid Prototyping==<br />
<br />
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. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 MakerBot.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerWare.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerBot ABS.jpg|x200px]]<br />
|-<br />
|'''Figure 4-a''' The MakerBot Replicator (2nd generation) we used to print our molds.<br />
|'''Figure 4-b''' 'Screenshot' of the MakerWare software we used to print our molds.<br />
|'''Figure 4-c''' A roll of ABS filament used by the 3D-printer.<br />
|}<br />
<br />
<br />
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).<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_comb_and_box.jpg|200px]]<br />
|[[File:ETH Zurich 2014 small grid.JPG|200px]]<br />
|[[File:ETH Zurich diffusion plate.JPG|200px]]<br />
|[[File:ETH Zurich 2014 96 well all connected.jpeg|200px]]<br />
|-<br />
|'''Figure 5-a''' Printed gel-comb and box. The box was autoclaved at 121 ⁰C. <br />
|'''Figure 5-b''' Printed millifluid grid with interconnected wells (edge length of 3 mm).<br />
|'''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).<br />
|'''Figure 5-d''' Printed 96-well plate, all wells (edge length of 5 mm) are connected.<br />
|}<br />
<br />
<html></article></html><br />
<br />
<html><article id='Preparation'></html><br />
<br />
==PDMS Chip Preparation==<br />
<br />
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].<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_first_mold_with_PDMS.jpg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion mold.JPG|200px]]<br />
|[[File:ETH Zurich 2014 final mold.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 final mold closeup.jpeg|200px]]<br />
|-<br />
|'''Figure 6-a''' The very first mold design. PDMS stuck between the wells while removing it. <br />
|'''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).<br />
|'''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). <br />
|'''Figure 6-d''' Close up of the final mold design. The separate layers are clearly visible ('additive' manufacturing).<br />
|}<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 broken chip.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 2 well diffusion chip upsidedown.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 PDMS diffusion chip final.jpeg|200px]]<br />
|[[File:ETH_Zurich_2014_final_chip_zoom.png|200px]]<br />
|-<br />
|'''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.<br />
|'''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).<br />
|'''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). <br />
|'''Figure 7-d''' Close up of the final PDMS chip. The channels are well defined and even small structures separated evenly from the mold.<br />
|}<br />
<html></article></html><br />
<br />
<html><article id='movies'></html><br />
<br />
==Time-Lapse Movies==<br />
<br />
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.<br />
<br />
<br />
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. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:100%; max-width: 650px; margin: auto;"<br />
|{{: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>}}<br />
|{{: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>}}<br />
|-<br />
|'''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.<br />
|'''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.<br />
|-<br />
|{{: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>}}<br />
|{{: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>}}<br />
|-<br />
|'''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, 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). ~1850x faster than real-time.<br />
|'''Video 4 Cell communication experiment with [ 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). ~3450x faster than real-time.<br />
|-<br />
|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>}}<br />
|-<br />
|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.<br />
|}<br />
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{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/lab/sequencesTeam:ETH Zurich/lab/sequences2014-10-18T01:04:25Z<p>Mbahls: /* Producer Constructs */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Sequences}}<br />
<html><article></html><br />
<br />
The plasmid sequences can be accessed by clicking on the plasmid name (e.g. [https://static.igem.org/mediawiki/2014/f/f6/ETH2014_piG0040.txt piG0040]) or the plasmid picture.<br />
<br />
Construct types:<br />
<br />
<br />
[[Team:ETH_Zurich/lab/sequences#Regulator_Constructs|Regulator Constructs]]<br />
<br />
[[Team:ETH_Zurich/lab/sequences#Producer_Constructs|Producer Constructs]]<br />
<br />
[[Team:ETH_Zurich/lab/sequences#Sensor Constructs|Sensor Constructs]]<br />
<br />
[[Team:ETH_Zurich/lab/sequences#BUFFER_Gate_Construct|BUFFER Gate Construct]]<br />
<br />
[[Team:ETH_Zurich/lab/sequences#Signal_Propagation_Construct|Signal Propagation Construct]]<br />
<br />
[[Team:ETH_Zurich/lab/sequences#Combined_Sensor_and_Producer_Constructs|Combined Sensor and Producer Constructs]]<br />
<br />
<br />
<br />
==Regulator Constructs==<br />
<br />
<br />
[https://static.igem.org/mediawiki/2014/f/f6/ETH2014_piG0040.txt piG0040]<br />
<br />
LasR is expressed under the strong constitutive promoter [http://parts.igem.org/Part:BBa_J23100 BBa_J23100] while the expression level is influenced by the RBS [http://parts.igem.org/Part:BBa_B0034 BBa_B0034]. LasR bound to 3OC12-HSL induces the expression of genes under the control of pLasR. The plasmid pBR322 and its derivatives have a copy number of 15 to 20<sup>[[Team:ETH_Zurich/project/references#refBolivar|[15]]]</sup>.<br />
<br />
[[File:ETH2014_piG0040_Map.png|link=https://static.igem.org/mediawiki/2014/f/f6/ETH2014_piG0040.txt]]<br />
<br />
<br />
[https://static.igem.org/mediawiki/2014/2/2c/ETH2014_piG0041.txt piG0041]<br />
<br />
LuxR is expressed under the strong constitutive promoter [http://parts.igem.org/Part:BBa_J23100 BBa_J23100] while the expression level is influenced by the RBS [http://parts.igem.org/Part:BBa_B0034 BBa_B0034]. LuxR bound to 3OC6-HSL induces the expression of genes under the control of pLuxR. The plasmid pBR322 and its derivatives have a copy number of 15 to 20<sup>[[Team:ETH_Zurich/project/references#refBolivar|[15]]]</sup>.<br />
<br />
<br />
[[File:ETH2014_piG0041_Map.png|link=https://static.igem.org/mediawiki/2014/2/2c/ETH2014_piG0041.txt]]<br />
<br />
<br />
[https://static.igem.org/mediawiki/2014/d/db/ETH2014_piG0042.txt piG0042]<br />
<br />
RhlR is expressed under the strong constitutive promoter [http://parts.igem.org/Part:BBa_J23100 BBa_J23100] while the expression level is influenced by the RBS [http://parts.igem.org/Part:BBa_B0034 BBa_B0034]. RhlR bound to C4-HSL induces the expression of genes under the control of pRhlR. The plasmid pBR322 and its derivatives have a copy number of 15 to 20<sup>[[Team:ETH_Zurich/project/references#refBolivar|[15]]]</sup>.<br />
<br />
<br />
[[File:ETH2014_piG0042_Map.png|link=https://static.igem.org/mediawiki/2014/d/db/ETH2014_piG0042.txt]]<br />
<br />
<br />
[https://static.igem.org/mediawiki/2014/c/ce/ETH2014_piG0042max.txt piG0042max]<br />
<br />
RhlR is expressed under the strong constitutive promoter [http://parts.igem.org/Part:BBa_J23100 BBa_J23100] while the expression level is influenced by an RBS optimised for RhlR ([https://salis.psu.edu/software/forward RBS calculator]). RhlR bound to C4-HSL induces the expression of genes under the control of pRhlR. The plasmid pBR322 and its derivatives have a copy number of 15 to 20<sup>[[Team:ETH_Zurich/project/references#refBolivar|[15]]]</sup>.<br />
<br />
<br />
[[File:ETH2014_piG0042max_Map.png|link=https://static.igem.org/mediawiki/2014/c/ce/ETH2014_piG0042max.txt]]<br />
<br />
<br />
[https://static.igem.org/mediawiki/2014/1/18/ETH2014_piG0046.txt piG0046]<br />
<br />
LuxR is expressed under the weak constitutive promoter [http://parts.igem.org/Part:BBa_J23109 BBa_J23109] while the expression level is influenced by the RBS [http://parts.igem.org/Part:BBa_B0034 BBa_B0034]. LuxR bound to 3OC6-HSL induces the expression of genes under the control of pLuxR. The plasmid pBR322 and its derivatives have a copy number of 15 to 20<sup>[[Team:ETH_Zurich/project/references#refBolivar|[15]]]</sup>.<br />
<br />
<br />
[[File:ETH2014_piG0046_Map.png|link=https://static.igem.org/mediawiki/2014/1/18/ETH2014_piG0046.txt]] <br />
<br />
<br />
[https://static.igem.org/mediawiki/2014/1/1b/ETH2014_piG0047.txt piG0047]<br />
<br />
LuxR is expressed under the medium strong constitutive promoter [http://parts.igem.org/Part:BBa_J23111 BBa_J23111 ] while the expression level is influenced by the RBS [http://parts.igem.org/Part:BBa_B0034 BBa_B0034]. LuxR bound to 3OC6-HSL induces the expression of genes under the control of pLuxR. The plasmid pBR322 and its derivatives have a copy number of 15 to 20<sup>[[Team:ETH_Zurich/project/references#refBolivar|[15]]]</sup>.<br />
<br />
<br />
[[File:ETH2014_piG0047_Map.png|link=https://static.igem.org/mediawiki/2014/1/1b/ETH2014_piG0047.txt]]<br />
<br />
<br />
<br />
<br />
==Producer Constructs==<br />
<br />
<br />
[https://static.igem.org/mediawiki/2014/4/43/ETH2014_piG0049.txt piG0049]<br />
<br />
LasI is expressed under the strong constitutive promoter [http://parts.igem.org/Part:BBa_J23100 BBa_J23100 ] while the expression level is influenced by the RBS [http://parts.igem.org/Part:BBa_B0034 BBa_B0034]. LasI produces the quorum sensing molecule 3OC12-HSL. The pBBR1 origin is present at a copy number of approximately 5, however, the origin is poorly characterized <sup>[[Team:ETH_Zurich/project/references#refLennen|[16]]]</sup>.<br />
<br />
<br />
[[File:ETH2014_piG0049_Map.png|link=https://static.igem.org/mediawiki/2014/4/43/ETH2014_piG0049.txt]]<br />
<br />
<br />
[https://static.igem.org/mediawiki/2014/b/bc/ETH2014_piG0049max.txt piG0049max]<br />
<br />
LasI is expressed under the strong constitutive promoter [http://parts.igem.org/Part:BBa_J23100 BBa_J23100 ] while the expression level is increased by an RBS optimised for LasI ([https://salis.psu.edu/software/forward RBS calculator]). LasI produces the quorum sensing molecule 3OC12-HSL. The pBBR1 origin is present at a copy number of approximately 5, however, the origin is poorly characterized <sup>[[Team:ETH_Zurich/project/references#refLennen|[16]]]</sup>.<br />
<br />
<br />
[[File:ETH2014_piG0049max_Map.png|link=https://static.igem.org/mediawiki/2014/b/bc/ETH2014_piG0049max.txt]]<br />
<br />
<br />
[https://static.igem.org/mediawiki/2014/4/49/ETH2014_piG0050.txt piG0050]<br />
<br />
LuxI is expressed under the strong constitutive promoter [http://parts.igem.org/Part:BBa_J23100 BBa_J23100 ] while the expression level is influenced by the RBS [http://parts.igem.org/Part:BBa_B0034 BBa_B0034]. LuxI produces the quorum sensing molecule 3OC6-HSL. The pBBR1 origin is present at a copy number of approximately 5, however, the origin is poorly characterized<sup>[[Team:ETH_Zurich/project/references#refLennen|[16]]]</sup>.<br />
<br />
[[File:ETH2014_piG0050_Map.png|link=https://static.igem.org/mediawiki/2014/4/49/ETH2014_piG0050.txt]]<br />
<br />
<br />
[https://static.igem.org/mediawiki/2014/5/50/ETH2014_piG0050max.txt piG0050max]<br />
<br />
LuxI is expressed under the strong constitutive promoter [http://parts.igem.org/Part:BBa_J23100 BBa_J23100 ] while the expression level is increased by an RBS optimised for LuxI ([https://salis.psu.edu/software/forward RBS calculator]). LuxI produces the quorum sensing molecule 3OC6-HSL. The pBBR1 origin is present at a copy number of approximately 5, however, the origin is poorly characterized<sup>[[Team:ETH_Zurich/project/references#refLennen|[16]]]</sup>.<br />
<br />
<br />
[[File:ETH2014_piG0050max_Map.png|link=https://static.igem.org/mediawiki/2014/5/50/ETH2014_piG0050max.txt]]<br />
<br />
<br />
[https://static.igem.org/mediawiki/2014/0/00/ETH2014_piG0051.txt piG0051]<br />
<br />
RhlI is expressed under the strong constitutive promoter [http://parts.igem.org/Part:BBa_J23100 BBa_J23100 ] while the expression level is influenced by the RBS [http://parts.igem.org/Part:BBa_B0034 BBa_B0034]. RhlI produces the quorum sensing molecule C4-HSL. The pBBR1 origin is present at a copy number of approximately 5, however, the origin is poorly characterized<sup>[[Team:ETH_Zurich/project/references#refLennen|[16]]]</sup>.<br />
<br />
[[File:ETH2014_piG0051_Map.png|link=https://static.igem.org/mediawiki/2014/0/00/ETH2014_piG0051.txt]]<br />
<br />
<br />
[https://static.igem.org/mediawiki/2014/9/94/ETH2014_piG0051max.txt piG0051max]<br />
<br />
RhlI is expressed under the strong constitutive promoter [http://parts.igem.org/Part:BBa_J23100 BBa_J23100 ] while the expression level is increased by an RBS optimised for RhlI ([https://salis.psu.edu/software/forward RBS calculator]). RhlI produces the quorum sensing molecule C4-HSL. The pBBR1 origin is present at a copy number of approximately 5, however, the origin is poorly characterized<sup>[[Team:ETH_Zurich/project/references#refLennen|[16]]]</sup>.<br />
<br />
[[File:ETH2014_piG0051max_Map.png|link=https://static.igem.org/mediawiki/2014/9/94/ETH2014_piG0051max.txt]]<br />
<br />
==Sensor Constructs==<br />
<br />
<br />
[https://static.igem.org/mediawiki/2014/a/a7/ETH2014_piG0058_sensor_plasRstRBS-sfGFP.txt piG0058]<br />
<br />
Expression of sfGFP is induced when LasR bound to 3OC12-HSL bind to pLasR. The p15A is present at a copy number of approximately 15 to 25<sup>[[Team:ETH_Zurich/project/references#refChang|[35]]]</sup>.<br />
<br />
[[File:ETH2014 piG0058 sensor plasRstRBS-sfGFP Map.png|link=https://static.igem.org/mediawiki/2014/a/a7/ETH2014_piG0058_sensor_plasRstRBS-sfGFP.txt]]<br />
<br />
[https://static.igem.org/mediawiki/2014/0/0f/ETH2014_piG0059_sensor_pluxRstRBS-sfGFP.txt piG0059]<br />
<br />
Expression of sfGFP is induced when LuxR bound to 3OC6-HSL bind to pLuxR. The p15A is present at a copy number of approximately 15 to 25<sup>[[Team:ETH_Zurich/project/references#refChang|[35]]]</sup>.<br />
<br />
[[File:ETH2014 piG0059 sensor pluxRstRBS-sfGFP Map.png|link=https://static.igem.org/mediawiki/2014/0/0f/ETH2014_piG0059_sensor_pluxRstRBS-sfGFP.txt]]<br />
<br />
[https://static.igem.org/mediawiki/2014/2/22/PiG0060_sensor_prhlRstRBS-sfGFP.txt piG0060]<br />
<br />
Expression of sfGFP is induced when RhlR bound to 4C-HSL bind to pRhlR. The p15A is present at a copy number of approximately 15 to 25<sup>[[Team:ETH_Zurich/project/references#refChang|[35]]]</sup>.<br />
<br />
[[File:ETH2014 piG0060 sensor prhlRstRBS-sfGFP Map.png|link=https://static.igem.org/mediawiki/2014/2/22/PiG0060_sensor_prhlRstRBS-sfGFP.txt]]<br />
<br />
[https://static.igem.org/mediawiki/2014/2/2c/PiG0062_sensor_pluxRcr12yRBS-sfGFP.txt piG0062]<br />
<br />
Expression of sfGFP is induced when LuxR bound to 3OC6-HSL bind to pLuxR. The cis-repressive element (crR12y) inhibits the translation of sfGFP, since the RBS is blocked by secondary structures of the mRNA. The p15A is present at a copy number of approximately 15 to 25<sup>[[Team:ETH_Zurich/project/references#refChang|[35]]]</sup>.<br />
<br />
[[File:ETH2014 piG0062 sensor pluxRcr12yRBS-sfGFP Map.png|link=https://static.igem.org/mediawiki/2014/2/2c/PiG0062_sensor_pluxRcr12yRBS-sfGFP.txt]]<br />
<br />
[https://static.igem.org/mediawiki/2014/d/da/ETH2014_piG0065_sensor_pluxRRR12y-sfGFP.txt piG0065]<br />
<br />
Expression of sfGFP is induced when LuxR bound to 3OC6-HSL bind to pLuxR. The cis-repressive element (crR12y) inhibits the translation of sfGFP, since the RBS is blocked by secondary structures of the mRNA. The transcript of the trans-activating element (taR12y) binds to the transcript of the cis-repressive element, hence the RBS is not blocked anymore. The two elements build a [https://2014.igem.org/Team:ETH_Zurich/expresults#Riboregulators riboregulator] that decreases leakiness of pLuxR. A biologically neutral spacer sequence was designed using the web application R2oDNA<sup>[[Team:ETH_Zurich/project/references#refR2oDNA|[34]]]</sup>. The p15A is present at a copy number of approximately 15 to 25<sup>[[Team:ETH_Zurich/project/references#refChang|[35]]]</sup>.<br />
<br />
[[File:ETH2014 piG0065 sensor pluxRRR12y-sfGFP Map.png|link=https://static.igem.org/mediawiki/2014/d/da/ETH2014_piG0065_sensor_pluxRRR12y-sfGFP.txt]]<br />
<br />
[https://static.igem.org/mediawiki/2014/2/24/ETH2014_piG0066_sensor_prhlRRR12-sfGFP.txt piG0066]<br />
<br />
Expression of sfGFP is induced when RhlR bound to 4C-HSL bind to pRhlR. The cis-repressive element (crR12) inhibits the translation of sfGFP, since the RBS is blocked by secondary structures of the mRNA. The transcript of the trans-activating element (taR12) binds to the transcript of the cis-repressive element, hence the RBS is not blocked anymore. The two elements build a [https://2014.igem.org/Team:ETH_Zurich/expresults#Riboregulators riboregulator] that decreases leakiness of pLuxR. A biologically neutral spacer sequence was designed using the web application R2oDNA<sup>[[Team:ETH_Zurich/project/references#refR2oDNA|[34]]]</sup>. The p15A is present at a copy number of approximately 15 to 25<sup>[[Team:ETH_Zurich/project/references#refChang|[35]]]</sup>.<br />
<br />
[[File:ETH2014 piG0066 sensor prhlRRR12-sfGFP Map.png|link=https://static.igem.org/mediawiki/2014/2/24/ETH2014_piG0066_sensor_prhlRRR12-sfGFP.txt]]<br />
<br />
[https://static.igem.org/mediawiki/2014/9/95/ETH2014_piG0109_pluxRRR12y-sfGFP_EcoRI-_XbaI-.txt piG0109]<br />
<br />
Expression of sfGFP is induced when LuxR bound to 3OC6-HSL bind to pLuxR. The cis-repressive element (crR12y) inhibits the translation of sfGFP, since the RBS is blocked by secondary structures of the mRNA. The transcript of the trans-activating element (taR12y) binds to the transcript of the cis-repressive element, hence the RBS is not blocked anymore. The two elements build a [https://2014.igem.org/Team:ETH_Zurich/expresults#Riboregulators riboregulator] that decreases leakiness of pLuxR. A biologically neutral spacer sequence was designed using the web application R2oDNA<sup>[[Team:ETH_Zurich/project/references#refR2oDNA|[34]]]</sup>. The p15A is present at a copy number of approximately 15 to 25<sup>[[Team:ETH_Zurich/project/references#refChang|[35]]]</sup>. PiG0109 is a derivate of piG0065 where the restriction sites EcoRI and XbaI have been removed. Thus, the two constructs slightly differ in the sequence of the 3'-end of the trans-activating element and in the sequence of the 5'-end of the cis-repressive element.<br />
<br />
[[File:ETH2014 piG0109 pluxRRR12y-sfGFP EcoRI- XbaI- Map.png|link=https://static.igem.org/mediawiki/2014/9/95/ETH2014_piG0109_pluxRRR12y-sfGFP_EcoRI-_XbaI-.txt]]<br />
<br />
[https://static.igem.org/mediawiki/2014/f/f9/ETH2014_piG0110_prhlRRR12-sfGFP_EcoRI-_XbaI-.txt piG0110]<br />
<br />
Expression of sfGFP is induced when RhlR bound to 4C-HSL bind to pRhlR. The cis-repressive element (crR12) inhibits the translation of sfGFP, since the RBS is blocked by secondary structures of the mRNA. The transcript of the trans-activating element (taR12) binds to the transcript of the cis-repressive element, hence the RBS is not blocked anymore. The two elements build a [https://2014.igem.org/Team:ETH_Zurich/expresults#Riboregulators riboregulator] that decreases leakiness of pLuxR. A biologically neutral spacer sequence was designed using the web application R2oDNA<sup>[[Team:ETH_Zurich/project/references#refR2oDNA|[34]]]</sup>. The p15A is present at a copy number of approximately 15 to 25<sup>[[Team:ETH_Zurich/project/references#refChang|[35]]]</sup>. PiG0110 is a derivate of piG0066 where the restriction sites EcoRI and XbaI have been removed. Thus, the two constructs slightly differ in the sequence of the 3'-end of the trans-activating element and in the sequence of the 5'-end of the cis-repressive element.<br />
<br />
[[File:ETH2014 piG0110 prhlRRR12-sfGFP EcoRI- XbaI- Map.png|link=https://static.igem.org/mediawiki/2014/f/f9/ETH2014_piG0110_prhlRRR12-sfGFP_EcoRI-_XbaI-.txt]]<br />
<br />
==BUFFER Gate Construct==<br />
<br />
[https://static.igem.org/mediawiki/2014/5/59/ETH2014_piG0067_logic_bxb1_BUFFER-sfGFP.txt piG0067]<br />
<br />
The directional terminator [http://parts.igem.org/Part:BBa_B0015 BBa_B0015] blocks transcription of sfGFP under the strong constitutive promoter [http://parts.igem.org/Part:BBa_J23100 BBa_J23100]. Only if the integrase Bxb1 flips B0015 between the attP and the attB sites, transcription of sfGFP is possible. The pBBR1 origin is present at a copy number of approximately 5, however, the origin is poorly characterized<sup>[[Team:ETH_Zurich/project/references#refLennen|[16]]]</sup><br />
<br />
<br />
[[File:ETH2014 piG0067 logic bxb1 BUFFER-sfGFP Map.png|link=https://static.igem.org/mediawiki/2014/5/59/ETH2014_piG0067_logic_bxb1_BUFFER-sfGFP.txt]]<br />
<br />
==Signal Propagation Construct==<br />
<br />
<br />
[https://static.igem.org/mediawiki/2014/1/1d/ETH2014_piG0071_sensor_pluxRRR12y_bxb1_mCherry.txt piG0071]<br />
<br />
Expression of the integrase Bxb1 and the fluorophore mCherry is induced when LuxR bound to 3OC6-HSL bind to pLuxR. The cis-repressive element (crR12y) inhibits the translation of Bxb1 and mCherry, since the RBS is blocked by secondary structures of the mRNA. The transcript of the trans-activating element (taR12y) binds to the transcript of the cis-repressive element, hence the RBS is not blocked anymore. The two elements build a [https://2014.igem.org/Team:ETH_Zurich/expresults#Riboregulators riboregulator] that decreases leakiness of pLuxR. A biologically neutral spacer sequence was designed using the web application R2oDNA<sup>[[Team:ETH_Zurich/project/references#refR2oDNA|[34]]]</sup>. The p15A is present at a copy number of approximately 15 to 25<sup>[[Team:ETH_Zurich/project/references#refChang|[35]]]</sup>.<br />
<br />
[[File:ETH2014 piG0071 sensor pluxRRR12y bxb1 mCherry Map.png|link=https://static.igem.org/mediawiki/2014/1/1d/ETH2014_piG0071_sensor_pluxRRR12y_bxb1_mCherry.txt]]<br />
<br />
<br />
<br />
<br />
==Combined Sensor and Producer Constructs==<br />
<br />
<br />
[https://static.igem.org/mediawiki/2014/f/f5/ETH2014_piG0096_pluxRRR12y-sfGFP-maxRBS-luxI.txt piG0096]<br />
<br />
Expression of sfGFP and LuxI is induced when LuxR bound to 3OC6-HSL bind to pLuxR. The cis-repressive element (crR12y) inhibits the translation of the succeeding gene, since the RBS is blocked by secondary structures of the mRNA. The transcript of the trans-activating element (taR12y) binds to the transcript of the cis-repressive element, hence the RBS is not blocked anymore. The two elements build a [https://2014.igem.org/Team:ETH_Zurich/expresults#Riboregulators riboregulator] that decreases leakiness of pLuxR. The expression level of LuxI is increased by an RBS optimised for LuxI ([https://salis.psu.edu/software/forward RBS calculator]). A biologically neutral spacer sequence was designed using the web application R2oDNA<sup>[[Team:ETH_Zurich/project/references#refR2oDNA|[34]]]</sup>. The p15A is present at a copy number of approximately 15 to 25<sup>[[Team:ETH_Zurich/project/references#refChang|[35]]]</sup>.<br />
<br />
[[File:ETH2014 piG0096 pluxRRR12y-sfGFP-maxRBS-luxI Map.png|link=https://static.igem.org/mediawiki/2014/f/f5/ETH2014_piG0096_pluxRRR12y-sfGFP-maxRBS-luxI.txt]]<br />
<br />
[https://static.igem.org/mediawiki/2014/f/fc/ETH2014_piG0097_pluxRRR12y-sfGFP-stRBS-luxI.txt piG0097]<br />
<br />
Expression of sfGFP and LuxI is induced when LuxR bound to 3OC6-HSL bind to pLuxR. The cis-repressive element (crR12y) inhibits the translation of the succeeding gene, since the RBS is blocked by secondary structures of the mRNA. The transcript of the trans-activating element (taR12y) binds to the transcript of the cis-repressive element, hence the RBS is not blocked anymore. The two elements build a [https://2014.igem.org/Team:ETH_Zurich/expresults#Riboregulators riboregulator] that decreases leakiness of pLuxR. The expression level of LuxI is influenced by the RBS [http://parts.igem.org/Part:BBa_B0034 BBa_B0034]. A biologically neutral spacer sequence was designed using the web application R2oDNA<sup>[[Team:ETH_Zurich/project/references#refR2oDNA|[34]]]</sup>. The p15A is present at a copy number of approximately 15 to 25<sup>[[Team:ETH_Zurich/project/references#refChang|[35]]]</sup>.<br />
<br />
[[File:ETH2014 piG0097 pluxRRR12y-sfGFP-stRBS-luxI Map.png|link=https://static.igem.org/mediawiki/2014/f/fc/ETH2014_piG0097_pluxRRR12y-sfGFP-stRBS-luxI.txt]]<br />
<br />
[https://static.igem.org/mediawiki/2014/6/69/ETH2014_piG0099_pluxstRBS-sfGFP-stRBS-luxI.txt piG0099]<br />
<br />
Expression of sfGFP and LuxI is induced when LuxR bound to 3OC6-HSL bind to pLuxR. The expression level of sfGFP and LuxI is influenced by the RBS [http://parts.igem.org/Part:BBa_B0034 BBa_B0034]. The p15A is present at a copy number of approximately 15 to 25<sup>[[Team:ETH_Zurich/project/references#refChang|[35]]]</sup>.<br />
<br />
[[File:ETH2014_piG0099_pluxstRBS-sfGFP-stRBS-luxI_Map.png|link=https://static.igem.org/mediawiki/2014/6/69/ETH2014_piG0099_pluxstRBS-sfGFP-stRBS-luxI.txt]]<br />
<br />
<br />
<html></article></html><br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/lab/chipTeam:ETH Zurich/lab/chip2014-10-18T01:00:17Z<p>Mbahls: /* Time-Lapse Movies */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Millifluidic Chip & Rapid Prototyping}}<br />
<br />
<html><article style='min-height:800px'></html><br />
==Overview==<br />
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).<br />
<br />
[[File:ETH Zurich Rule 6.PNG|300px|thumb|left|'''Figure 1''' '''Classical grid from cellular automata theory''' (ON state=back, OFF state=white).]]<br />
[[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).]]<br />
<br />
<br />
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.<br />
<br><br />
<br><br />
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.<br />
<br />
<html></article></html><br />
<br />
<br />
<html><article></html><br />
<br />
==Mold Design and 3D Print Exchange==<br />
<br />
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>. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_mesh_2_20140826.jpg|200px]]<br />
|[[File:ETH Zurich 2014 final mold model 2.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion plate model.jpeg|200px]]<br />
|-<br />
|'''Figure 3-a''' The first design for a gel-comb, a mold for a millifluidic PDMS chip and a corresponding box for the mold.<br />
|'''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. <br />
|'''Figure 3-c''' A design for a 96-well plate with connected wells, which allows automated measurements in a plate reader.<br />
|}<br />
<br />
<br />
'''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.<br />
'''<br />
<html></article></html><br />
<br />
<html><article></html><br />
<br />
==3D-Printing and Rapid Prototyping==<br />
<br />
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. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 MakerBot.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerWare.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerBot ABS.jpg|x200px]]<br />
|-<br />
|'''Figure 4-a''' The MakerBot Replicator (2nd generation) we used to print our molds.<br />
|'''Figure 4-b''' 'Screenshot' of the MakerWare software we used to print our molds.<br />
|'''Figure 4-c''' A roll of ABS filament used by the 3D-printer.<br />
|}<br />
<br />
<br />
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).<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_comb_and_box.jpg|200px]]<br />
|[[File:ETH Zurich 2014 small grid.JPG|200px]]<br />
|[[File:ETH Zurich diffusion plate.JPG|200px]]<br />
|[[File:ETH Zurich 2014 96 well all connected.jpeg|200px]]<br />
|-<br />
|'''Figure 5-a''' Printed gel-comb and box. The box was autoclaved at 121 ⁰C. <br />
|'''Figure 5-b''' Printed millifluid grid with interconnected wells (edge length of 3 mm).<br />
|'''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).<br />
|'''Figure 5-d''' Printed 96-well plate, all wells (edge length of 5 mm) are connected.<br />
|}<br />
<br />
<html></article></html><br />
<br />
<html><article id='Preparation'></html><br />
<br />
==PDMS Chip Preparation==<br />
<br />
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].<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_first_mold_with_PDMS.jpg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion mold.JPG|200px]]<br />
|[[File:ETH Zurich 2014 final mold.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 final mold closeup.jpeg|200px]]<br />
|-<br />
|'''Figure 6-a''' The very first mold design. PDMS stuck between the wells while removing it. <br />
|'''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).<br />
|'''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). <br />
|'''Figure 6-d''' Close up of the final mold design. The separate layers are clearly visible ('additive' manufacturing).<br />
|}<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 broken chip.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 2 well diffusion chip upsidedown.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 PDMS diffusion chip final.jpeg|200px]]<br />
|[[File:ETH_Zurich_2014_final_chip_zoom.png|200px]]<br />
|-<br />
|'''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.<br />
|'''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).<br />
|'''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). <br />
|'''Figure 7-d''' Close up of the final PDMS chip. The channels are well defined and even small structures separated evenly from the mold.<br />
|}<br />
<html></article></html><br />
<br />
<html><article id='movies'></html><br />
<br />
==Time-Lapse Movies==<br />
<br />
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.<br />
<br />
<br />
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. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:100%; max-width: 650px; margin: auto;"<br />
|{{: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>}}<br />
|{{: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>}}<br />
|-<br />
|'''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 [ LB agar]. About 5 μL ink were added in a punched whole on one side of the two wells. ~4500x faster than real-time.<br />
|'''Video 2 Diffusion experiment with liquid cultures.''' The wells of the PDMS chip were filled with LB medium, separated by solidified 2% agarose in the channel. The bottom well contained 3OC6-HSL, the top well ''E. coli'' cells with sfGFP under the control of pLux. ~4500x faster than real-time.<br />
|-<br />
|{{: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>}}<br />
|{{: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>}}<br />
|-<br />
|'''Video 3 Diffusion experiment with 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, the top well ''E. coli'' cells with sfGFP under the control of pLux confined in alginate beads (d=3 mm). ~1850x faster than real-time.<br />
|'''Video 4 Cell communication experiment with alginate beads in defined liquid medium.''' The wells of the PDMS chip were filled with CB medium. The bottom well contained ''E. coli'' cells expressing LuxI, which catalyzes the production of 3OC6-HSL; the top well contained ''E. coli'' cells with sfGFP under the control of pLux, All cells were confined in alginate beads (d=3 mm). ~3450x faster than real-time.<br />
|-<br />
|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>}}<br />
|-<br />
|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.<br />
|}<br />
<br />
<br />
<html></article></html><br />
<br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/lab/chipTeam:ETH Zurich/lab/chip2014-10-18T00:59:00Z<p>Mbahls: /* Time-Lapse Movies */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Millifluidic Chip & Rapid Prototyping}}<br />
<br />
<html><article style='min-height:800px'></html><br />
==Overview==<br />
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).<br />
<br />
[[File:ETH Zurich Rule 6.PNG|300px|thumb|left|'''Figure 1''' '''Classical grid from cellular automata theory''' (ON state=back, OFF state=white).]]<br />
[[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).]]<br />
<br />
<br />
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.<br />
<br><br />
<br><br />
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.<br />
<br />
<html></article></html><br />
<br />
<br />
<html><article></html><br />
<br />
==Mold Design and 3D Print Exchange==<br />
<br />
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>. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_mesh_2_20140826.jpg|200px]]<br />
|[[File:ETH Zurich 2014 final mold model 2.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion plate model.jpeg|200px]]<br />
|-<br />
|'''Figure 3-a''' The first design for a gel-comb, a mold for a millifluidic PDMS chip and a corresponding box for the mold.<br />
|'''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. <br />
|'''Figure 3-c''' A design for a 96-well plate with connected wells, which allows automated measurements in a plate reader.<br />
|}<br />
<br />
<br />
'''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.<br />
'''<br />
<html></article></html><br />
<br />
<html><article></html><br />
<br />
==3D-Printing and Rapid Prototyping==<br />
<br />
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. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 MakerBot.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerWare.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerBot ABS.jpg|x200px]]<br />
|-<br />
|'''Figure 4-a''' The MakerBot Replicator (2nd generation) we used to print our molds.<br />
|'''Figure 4-b''' 'Screenshot' of the MakerWare software we used to print our molds.<br />
|'''Figure 4-c''' A roll of ABS filament used by the 3D-printer.<br />
|}<br />
<br />
<br />
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).<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_comb_and_box.jpg|200px]]<br />
|[[File:ETH Zurich 2014 small grid.JPG|200px]]<br />
|[[File:ETH Zurich diffusion plate.JPG|200px]]<br />
|[[File:ETH Zurich 2014 96 well all connected.jpeg|200px]]<br />
|-<br />
|'''Figure 5-a''' Printed gel-comb and box. The box was autoclaved at 121 ⁰C. <br />
|'''Figure 5-b''' Printed millifluid grid with interconnected wells (edge length of 3 mm).<br />
|'''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).<br />
|'''Figure 5-d''' Printed 96-well plate, all wells (edge length of 5 mm) are connected.<br />
|}<br />
<br />
<html></article></html><br />
<br />
<html><article id='Preparation'></html><br />
<br />
==PDMS Chip Preparation==<br />
<br />
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].<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_first_mold_with_PDMS.jpg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion mold.JPG|200px]]<br />
|[[File:ETH Zurich 2014 final mold.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 final mold closeup.jpeg|200px]]<br />
|-<br />
|'''Figure 6-a''' The very first mold design. PDMS stuck between the wells while removing it. <br />
|'''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).<br />
|'''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). <br />
|'''Figure 6-d''' Close up of the final mold design. The separate layers are clearly visible ('additive' manufacturing).<br />
|}<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 broken chip.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 2 well diffusion chip upsidedown.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 PDMS diffusion chip final.jpeg|200px]]<br />
|[[File:ETH_Zurich_2014_final_chip_zoom.png|200px]]<br />
|-<br />
|'''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.<br />
|'''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).<br />
|'''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). <br />
|'''Figure 7-d''' Close up of the final PDMS chip. The channels are well defined and even small structures separated evenly from the mold.<br />
|}<br />
<html></article></html><br />
<br />
<html><article id='movies'></html><br />
<br />
==Time-Lapse Movies==<br />
<br />
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.<br />
<br />
<br />
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. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:100%; max-width: 650px; margin: auto;"<br />
|{{: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>}}<br />
|{{: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>}}<br />
|-<br />
|'''Video 1 The very first diffusion experiment with fluorescent highlighter ink (pyranine).''' The wells of the PDMS chip were filled with LB agar. About 5 μL ink were added in a punched whole on one side of the two wells. ~4500x faster than real-time.<br />
|'''Video 2 Diffusion experiment with liquid cultures.''' The wells of the PDMS chip were filled with LB medium, separated by solidified 2% agarose in the channel. The bottom well contained 3OC6-HSL, the top well ''E. coli'' cells with sfGFP under the control of pLux. ~4500x faster than real-time.<br />
|-<br />
|{{: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>}}<br />
|{{: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>}}<br />
|-<br />
|'''Video 3 Diffusion experiment with 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, the top well ''E. coli'' cells with sfGFP under the control of pLux confined in alginate beads (d=3 mm). ~1850x faster than real-time.<br />
|'''Video 4 Cell communication experiment with alginate beads in defined liquid medium.''' The wells of the PDMS chip were filled with CB medium. The bottom well contained ''E. coli'' cells expressing LuxI, which catalyzes the production of 3OC6-HSL; the top well contained ''E. coli'' cells with sfGFP under the control of pLux, All cells were confined in alginate beads (d=3 mm). ~3450x faster than real-time.<br />
|-<br />
|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>}}<br />
|-<br />
|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.<br />
|}<br />
<br />
<br />
<html></article></html><br />
<br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/lab/chipTeam:ETH Zurich/lab/chip2014-10-18T00:57:57Z<p>Mbahls: </p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Millifluidic Chip & Rapid Prototyping}}<br />
<br />
<html><article style='min-height:800px'></html><br />
==Overview==<br />
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).<br />
<br />
[[File:ETH Zurich Rule 6.PNG|300px|thumb|left|'''Figure 1''' '''Classical grid from cellular automata theory''' (ON state=back, OFF state=white).]]<br />
[[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).]]<br />
<br />
<br />
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.<br />
<br><br />
<br><br />
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.<br />
<br />
<html></article></html><br />
<br />
<br />
<html><article></html><br />
<br />
==Mold Design and 3D Print Exchange==<br />
<br />
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>. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_mesh_2_20140826.jpg|200px]]<br />
|[[File:ETH Zurich 2014 final mold model 2.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion plate model.jpeg|200px]]<br />
|-<br />
|'''Figure 3-a''' The first design for a gel-comb, a mold for a millifluidic PDMS chip and a corresponding box for the mold.<br />
|'''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. <br />
|'''Figure 3-c''' A design for a 96-well plate with connected wells, which allows automated measurements in a plate reader.<br />
|}<br />
<br />
<br />
'''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.<br />
'''<br />
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<br />
<html><article></html><br />
<br />
==3D-Printing and Rapid Prototyping==<br />
<br />
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. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 MakerBot.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerWare.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerBot ABS.jpg|x200px]]<br />
|-<br />
|'''Figure 4-a''' The MakerBot Replicator (2nd generation) we used to print our molds.<br />
|'''Figure 4-b''' 'Screenshot' of the MakerWare software we used to print our molds.<br />
|'''Figure 4-c''' A roll of ABS filament used by the 3D-printer.<br />
|}<br />
<br />
<br />
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).<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_comb_and_box.jpg|200px]]<br />
|[[File:ETH Zurich 2014 small grid.JPG|200px]]<br />
|[[File:ETH Zurich diffusion plate.JPG|200px]]<br />
|[[File:ETH Zurich 2014 96 well all connected.jpeg|200px]]<br />
|-<br />
|'''Figure 5-a''' Printed gel-comb and box. The box was autoclaved at 121 ⁰C. <br />
|'''Figure 5-b''' Printed millifluid grid with interconnected wells (edge length of 3 mm).<br />
|'''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).<br />
|'''Figure 5-d''' Printed 96-well plate, all wells (edge length of 5 mm) are connected.<br />
|}<br />
<br />
<html></article></html><br />
<br />
<html><article id='Preparation'></html><br />
<br />
==PDMS Chip Preparation==<br />
<br />
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].<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_first_mold_with_PDMS.jpg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion mold.JPG|200px]]<br />
|[[File:ETH Zurich 2014 final mold.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 final mold closeup.jpeg|200px]]<br />
|-<br />
|'''Figure 6-a''' The very first mold design. PDMS stuck between the wells while removing it. <br />
|'''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).<br />
|'''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). <br />
|'''Figure 6-d''' Close up of the final mold design. The separate layers are clearly visible ('additive' manufacturing).<br />
|}<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 broken chip.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 2 well diffusion chip upsidedown.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 PDMS diffusion chip final.jpeg|200px]]<br />
|[[File:ETH_Zurich_2014_final_chip_zoom.png|200px]]<br />
|-<br />
|'''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.<br />
|'''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).<br />
|'''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). <br />
|'''Figure 7-d''' Close up of the final PDMS chip. The channels are well defined and even small structures separated evenly from the mold.<br />
|}<br />
<html></article></html><br />
<br />
<html><article id='movies'></html><br />
<br />
==Time-Lapse Movies==<br />
<br />
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.<br />
<br />
<br />
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. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:100%; max-width: 650px; margin: auto;"<br />
|{{: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>}}<br />
|{{: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>}}<br />
|-<br />
|'''Video 1 The very first diffusion experiment with fluorescent highlighter ink (pyranine).''' The wells of the PDMS chip were filled with LB agar. About 5 μL ink were added in a punched whole on one side of the two wells. ~4500x faster than real-time.<br />
|'''Video 2 Diffusion experiment with liquid cultures.''' The wells of the PDMS chip were filled with LB medium, separated by solidified 2% agarose in the channel. The bottom well contained 3OC6-HSL, the top well ''E. coli'' cells with sfGFP under the control of pLux. ~4500x faster than real-time.<br />
|-<br />
|{{: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>}}<br />
|{{: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>}}<br />
|-<br />
|'''Video 3 Diffusion experiment with 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, the top well ''E. coli'' cells with sfGFP under the control of pLux confined in alginate beads (d=3 mm). ~1850x faster than real-time.<br />
|'''Video 4 Cell communication experiment with alginate beads in defined liquid medium.''' The wells of the PDMS chip were filled with CB medium. The bottom well contained ''E. coli'' cells expressing LuxI, which catalyzes the production of 3OC6-HSL; the top well contained ''E. coli'' cells with sfGFP under the control of pLux, All cells were confined in alginate beads (d=3 mm). ~3450x faster than real-time.<br />
|-<br />
|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>}}<br />
|-<br />
|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.<br />
|}<br />
<br />
<br />
<html></article></html><br />
<br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/lab/chipTeam:ETH Zurich/lab/chip2014-10-18T00:49:15Z<p>Mbahls: </p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Millifluidic Chip & Rapid Prototyping}}<br />
<br />
<html><article style='min-height:800px'></html><br />
==Overview==<br />
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).<br />
<br />
[[File:ETH Zurich Rule 6.PNG|300px|thumb|left|'''Figure 1''' '''Classical grid from cellular automata theory''' (ON state=back, OFF state=white).]]<br />
[[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).]]<br />
<br />
<br />
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.<br />
<br><br />
<br><br />
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.<br />
<br />
<html></article></html><br />
<br />
<br />
<html><article></html><br />
<br />
==Mold Design and 3D Print Exchange==<br />
<br />
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>. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_mesh_2_20140826.jpg|200px]]<br />
|[[File:ETH Zurich 2014 final mold model 2.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion plate model.jpeg|200px]]<br />
|-<br />
|'''Figure 3-a''' The first design for a gel-comb, a mold for a millifluidic PDMS chip and a corresponding box for the mold.<br />
|'''Figure 3-b''' The final mold design for our millifluid PDMS chip used for cell-to-cell communication experiments. <br />
|'''Figure 3-c''' A design for a 96-well plate with connected wells, which allows automated measurements in a plate reader.<br />
|}<br />
<br />
<br />
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.<br />
<br />
<html></article></html><br />
<br />
<html><article></html><br />
<br />
==3D-Printing and Rapid Prototyping==<br />
<br />
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. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 MakerBot.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerWare.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerBot ABS.jpg|x200px]]<br />
|-<br />
|'''Figure 4-a''' The MakerBot Replicator (2nd generation) we used to print our molds.<br />
|'''Figure 4-b''' 'Screenshot' of the MakerWare software we used to print our molds.<br />
|'''Figure 4-c''' A roll of ABS filament used by the 3D-printer.<br />
|}<br />
<br />
<br />
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).<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_comb_and_box.jpg|200px]]<br />
|[[File:ETH Zurich 2014 small grid.JPG|200px]]<br />
|[[File:ETH Zurich diffusion plate.JPG|200px]]<br />
|[[File:ETH Zurich 2014 96 well all connected.jpeg|200px]]<br />
|-<br />
|'''Figure 5-a''' Printed gel-comb and box. The box was autoclaved at 121 ⁰C. <br />
|'''Figure 5-b''' Printed millifluid grid with interconnected wells (edge length of 3 mm).<br />
|'''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).<br />
|'''Figure 5-d''' Printed 96-well plate, all wells (edge length of 5 mm) are connected.<br />
|}<br />
<br />
<html></article></html><br />
<br />
<html><article id='Preparation'></html><br />
<br />
==PDMS Chip Preparation==<br />
<br />
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].<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_first_mold_with_PDMS.jpg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion mold.JPG|200px]]<br />
|[[File:ETH Zurich 2014 final mold.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 final mold closeup.jpeg|200px]]<br />
|-<br />
|'''Figure 6-a''' The very first mold design. PDMS stuck between the wells while removing it. <br />
|'''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).<br />
|'''Figure 6-c''' The final mold design for cell-to-cell communication experiments (edge length of 5 mm, channel length of 3 mm). <br />
|'''Figure 6-d''' Close up of the final mold design. The separate layers are clearly visible ('additive' manufacturing).<br />
|}<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 broken chip.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 2 well diffusion chip upsidedown.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 PDMS diffusion chip final.jpeg|200px]]<br />
|[[File:ETH_Zurich_2014_final_chip_zoom.png|200px]]<br />
|-<br />
|'''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.<br />
|'''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).<br />
|'''Figure 7-c''' The final PDMS chip for cell-to-cell communication experiments (edge length of 5 mm, channel length of 3 mm). <br />
|'''Figure 7-d''' Close up of the final PDMS chip. The channels are well defined and even small structures separated evenly from the mold.<br />
|}<br />
<html></article></html><br />
<br />
<html><article id='movies'></html><br />
<br />
==Time-Lapse Movies==<br />
<br />
Below you find an overview of 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 (see video 1). 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. 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.<br />
<br />
<br />
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. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:100%; max-width: 650px; margin: auto;"<br />
|{{: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>}}<br />
|{{: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>}}<br />
|-<br />
|'''Video 1 The very first diffusion experiment with fluorescent highlighter ink (pyranine).''' The wells of the PDMS chip were filled with LB agar. About 5 μL ink were added in a punched whole on one side of the two wells. ~4500x faster than real-time.<br />
|'''Video 2 Diffusion experiment with liquid cultures.''' The wells of the PDMS chip were filled with LB medium, separated by solidified 2% agarose in the channel. The bottom well contained 3OC6-HSL, the top well ''E. coli'' cells with sfGFP under the control of pLux. ~4500x faster than real-time.<br />
|-<br />
|{{: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>}}<br />
|{{: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>}}<br />
|-<br />
|'''Video 3 Diffusion experiment with 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, the top well ''E. coli'' cells with sfGFP under the control of pLux confined in alginate beads (d=3 mm). ~1850x faster than real-time.<br />
|'''Video 4 Cell communication experiment with alginate beads in defined liquid medium.''' The wells of the PDMS chip were filled with CB medium. The bottom well contained ''E. coli'' cells expressing LuxI, which catalyzes the production of 3OC6-HSL; the top well contained ''E. coli'' cells with sfGFP under the control of pLux, All cells were confined in alginate beads (d=3 mm). ~3450x faster than real-time.<br />
|-<br />
|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>}}<br />
|-<br />
|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.<br />
|}<br />
<br />
<br />
<html></article></html><br />
<br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/lab/chipTeam:ETH Zurich/lab/chip2014-10-18T00:45:11Z<p>Mbahls: /* PDMS Chip Preparation */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Millifluidic Chip & Rapid Prototyping}}<br />
<br />
<html><article style='min-height:800px'></html><br />
==Overview==<br />
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).<br />
<br />
[[File:ETH Zurich Rule 6.PNG|300px|thumb|left|'''Figure 1''' '''Classical grid from cellular automata theory''' (ON state=back, OFF state=white).]]<br />
[[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).]]<br />
<br />
<br />
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.<br />
<br><br />
<br><br />
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.<br />
<br />
<html></article></html><br />
<br />
<br />
<html><article></html><br />
<br />
==Mold Design and 3D Print Exchange==<br />
<br />
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>. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_mesh_2_20140826.jpg|200px]]<br />
|[[File:ETH Zurich 2014 final mold model 2.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion plate model.jpeg|200px]]<br />
|-<br />
|'''Figure 3-a''' The first design for a gel-comb, a mold for a millifluidic PDMS chip and a corresponding box for the mold.<br />
|'''Figure 3-b''' The final mold design for our millifluid PDMS chip used for cell-to-cell communication experiments. <br />
|'''Figure 3-c''' A design for a 96-well plate with connected wells, which allows automated measurements in a plate reader.<br />
|}<br />
<br />
<br />
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.<br />
<br />
<html></article></html><br />
<br />
<html><article></html><br />
<br />
==3D-Printing and Rapid Prototyping==<br />
<br />
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. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 MakerBot.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerWare.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerBot ABS.jpg|x200px]]<br />
|-<br />
|'''Figure 4-a''' The MakerBot Replicator (2nd generation) we used to print our molds.<br />
|'''Figure 4-b''' 'Screenshot' of the MakerWare software we used to print our molds.<br />
|'''Figure 4-c''' A roll of ABS filament used by the 3D-printer.<br />
|}<br />
<br />
<br />
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).<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_comb_and_box.jpg|200px]]<br />
|[[File:ETH Zurich 2014 small grid.JPG|200px]]<br />
|[[File:ETH Zurich diffusion plate.JPG|200px]]<br />
|[[File:ETH Zurich 2014 96 well all connected.jpeg|200px]]<br />
|-<br />
|'''Figure 5-a''' Printed gel-comb and box. The box was autoclaved at 121 ⁰C. <br />
|'''Figure 5-b''' Printed millifluid grid with interconnected wells (edge length of 3 mm).<br />
|'''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).<br />
|'''Figure 5-d''' Printed 96-well plate, all wells (edge length of 5 mm) are connected.<br />
|}<br />
<br />
<html></article></html><br />
<br />
<html><article id='Preparation'></html><br />
<br />
==PDMS Chip Preparation==<br />
<br />
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].<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_first_mold_with_PDMS.jpg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion mold.JPG|200px]]<br />
|[[File:ETH Zurich 2014 final mold.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 final mold closeup.jpeg|200px]]<br />
|-<br />
|'''Figure 6-a''' The very first mold design. PDMS stuck between the wells while removing it. <br />
|'''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).<br />
|'''Figure 6-c''' The final mold design for cell-to-cell communication experiments (edge length of 5 mm, channel length of 3 mm). <br />
|'''Figure 6-d''' Close up of the final mold design. The separate layers are clearly visible ('additive' manufacturing).<br />
|}<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 broken chip.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 2 well diffusion chip upsidedown.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 PDMS diffusion chip final.jpeg|200px]]<br />
|[[File:ETH_Zurich_2014_final_chip_zoom.png|200px]]<br />
|-<br />
|'''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.<br />
|'''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).<br />
|'''Figure 7-c''' The final PDMS chip for cell-to-cell communication experiments (edge length of 5 mm, channel length of 3 mm). <br />
|'''Figure 7-d''' Close up of the final PDMS chip. The channels are well defined and even small structures separated evenly from the mold.<br />
|}<br />
<html></article></html><br />
<br />
<html><article id='movies'></html><br />
<br />
==Time-Lapse Movies==<br />
<br />
Below you find an overview of 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 (see video 1). 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. 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.<br />
<br />
<br />
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. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:100%; max-width: 650px; margin: auto;"<br />
|{{: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>}}<br />
|{{: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>}}<br />
|-<br />
|'''Video 1 The very first diffusion experiment with fluorescent highlighter ink (pyranine).''' The wells of the PDMS chip were filled with LB agar. About 5 μL ink were added in a punched whole on one side of the two wells. ~4500x faster than real-time.<br />
|'''Video 2 Diffusion experiment with liquid cultures.''' The wells of the PDMS chip were filled with LB medium, separated by solidified 2% agarose in the channel. The bottom well contained 3OC6-HSL, the top well ''E. coli'' cells with sfGFP under the control of pLux. ~4500x faster than real-time.<br />
|-<br />
|{{: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>}}<br />
|{{: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>}}<br />
|-<br />
|'''Video 3 Diffusion experiment with 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, the top well ''E. coli'' cells with sfGFP under the control of pLux confined in alginate beads (d=3 mm). ~1850x faster than real-time.<br />
|'''Video 4 Cell communication experiment with alginate beads in defined liquid medium.''' The wells of the PDMS chip were filled with CB medium. The bottom well contained ''E. coli'' cells expressing LuxI, which catalyzes the production of 3OC6-HSL; the top well contained ''E. coli'' cells with sfGFP under the control of pLux, All cells were confined in alginate beads (d=3 mm). ~3450x faster than real-time.<br />
|-<br />
|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>}}<br />
|-<br />
|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.<br />
|}<br />
<br />
<br />
<html></article></html><br />
<br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/lab/chipTeam:ETH Zurich/lab/chip2014-10-18T00:44:01Z<p>Mbahls: /* PDMS Chip Preparation */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Millifluidic Chip & Rapid Prototyping}}<br />
<br />
<html><article style='min-height:800px'></html><br />
==Overview==<br />
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).<br />
<br />
[[File:ETH Zurich Rule 6.PNG|300px|thumb|left|'''Figure 1''' '''Classical grid from cellular automata theory''' (ON state=back, OFF state=white).]]<br />
[[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).]]<br />
<br />
<br />
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.<br />
<br><br />
<br><br />
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.<br />
<br />
<html></article></html><br />
<br />
<br />
<html><article></html><br />
<br />
==Mold Design and 3D Print Exchange==<br />
<br />
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>. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_mesh_2_20140826.jpg|200px]]<br />
|[[File:ETH Zurich 2014 final mold model 2.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion plate model.jpeg|200px]]<br />
|-<br />
|'''Figure 3-a''' The first design for a gel-comb, a mold for a millifluidic PDMS chip and a corresponding box for the mold.<br />
|'''Figure 3-b''' The final mold design for our millifluid PDMS chip used for cell-to-cell communication experiments. <br />
|'''Figure 3-c''' A design for a 96-well plate with connected wells, which allows automated measurements in a plate reader.<br />
|}<br />
<br />
<br />
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.<br />
<br />
<html></article></html><br />
<br />
<html><article></html><br />
<br />
==3D-Printing and Rapid Prototyping==<br />
<br />
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. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 MakerBot.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerWare.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerBot ABS.jpg|x200px]]<br />
|-<br />
|'''Figure 4-a''' The MakerBot Replicator (2nd generation) we used to print our molds.<br />
|'''Figure 4-b''' 'Screenshot' of the MakerWare software we used to print our molds.<br />
|'''Figure 4-c''' A roll of ABS filament used by the 3D-printer.<br />
|}<br />
<br />
<br />
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).<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_comb_and_box.jpg|200px]]<br />
|[[File:ETH Zurich 2014 small grid.JPG|200px]]<br />
|[[File:ETH Zurich diffusion plate.JPG|200px]]<br />
|[[File:ETH Zurich 2014 96 well all connected.jpeg|200px]]<br />
|-<br />
|'''Figure 5-a''' Printed gel-comb and box. The box was autoclaved at 121 ⁰C. <br />
|'''Figure 5-b''' Printed millifluid grid with interconnected wells (edge length of 3 mm).<br />
|'''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).<br />
|'''Figure 5-d''' Printed 96-well plate, all wells (edge length of 5 mm) are connected.<br />
|}<br />
<br />
<html></article></html><br />
<br />
<html><article id='Preparation'></html><br />
<br />
==PDMS Chip Preparation==<br />
<br />
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). The 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]. All the mold shown below were at least used once before the pictures were taken.<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_first_mold_with_PDMS.jpg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion mold.JPG|200px]]<br />
|[[File:ETH Zurich 2014 final mold.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 final mold closeup.jpeg|200px]]<br />
|-<br />
|'''Figure 6-a''' The very first mold design. PDMS stuck between the wells while removing it. <br />
|'''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).<br />
|'''Figure 6-c''' The final mold design for cell-to-cell communication experiments (edge length of 5 mm, channel length of 3 mm). <br />
|'''Figure 6-d''' Close up of the final mold design. The separate layers are clearly visible ('additive' manufacturing).<br />
|}<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 broken chip.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 2 well diffusion chip upsidedown.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 PDMS diffusion chip final.jpeg|200px]]<br />
|[[File:ETH_Zurich_2014_final_chip_zoom.png|200px]]<br />
|-<br />
|'''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.<br />
|'''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).<br />
|'''Figure 7-c''' The final PDMS chip for cell-to-cell communication experiments (edge length of 5 mm, channel length of 3 mm). <br />
|'''Figure 7-d''' Close up of the final PDMS chip. The channels are well defined and even small structures separated evenly from the mold.<br />
|}<br />
<html></article></html><br />
<br />
<html><article id='movies'></html><br />
<br />
==Time-Lapse Movies==<br />
<br />
Below you find an overview of 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 (see video 1). 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. 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.<br />
<br />
<br />
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. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:100%; max-width: 650px; margin: auto;"<br />
|{{: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>}}<br />
|{{: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>}}<br />
|-<br />
|'''Video 1 The very first diffusion experiment with fluorescent highlighter ink (pyranine).''' The wells of the PDMS chip were filled with LB agar. About 5 μL ink were added in a punched whole on one side of the two wells. ~4500x faster than real-time.<br />
|'''Video 2 Diffusion experiment with liquid cultures.''' The wells of the PDMS chip were filled with LB medium, separated by solidified 2% agarose in the channel. The bottom well contained 3OC6-HSL, the top well ''E. coli'' cells with sfGFP under the control of pLux. ~4500x faster than real-time.<br />
|-<br />
|{{: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>}}<br />
|{{: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>}}<br />
|-<br />
|'''Video 3 Diffusion experiment with 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, the top well ''E. coli'' cells with sfGFP under the control of pLux confined in alginate beads (d=3 mm). ~1850x faster than real-time.<br />
|'''Video 4 Cell communication experiment with alginate beads in defined liquid medium.''' The wells of the PDMS chip were filled with CB medium. The bottom well contained ''E. coli'' cells expressing LuxI, which catalyzes the production of 3OC6-HSL; the top well contained ''E. coli'' cells with sfGFP under the control of pLux, All cells were confined in alginate beads (d=3 mm). ~3450x faster than real-time.<br />
|-<br />
|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>}}<br />
|-<br />
|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.<br />
|}<br />
<br />
<br />
<html></article></html><br />
<br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/lab/chipTeam:ETH Zurich/lab/chip2014-10-18T00:43:50Z<p>Mbahls: /* PDMS Chip Preparation */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Millifluidic Chip & Rapid Prototyping}}<br />
<br />
<html><article style='min-height:800px'></html><br />
==Overview==<br />
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).<br />
<br />
[[File:ETH Zurich Rule 6.PNG|300px|thumb|left|'''Figure 1''' '''Classical grid from cellular automata theory''' (ON state=back, OFF state=white).]]<br />
[[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).]]<br />
<br />
<br />
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.<br />
<br><br />
<br><br />
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.<br />
<br />
<html></article></html><br />
<br />
<br />
<html><article></html><br />
<br />
==Mold Design and 3D Print Exchange==<br />
<br />
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>. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_mesh_2_20140826.jpg|200px]]<br />
|[[File:ETH Zurich 2014 final mold model 2.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion plate model.jpeg|200px]]<br />
|-<br />
|'''Figure 3-a''' The first design for a gel-comb, a mold for a millifluidic PDMS chip and a corresponding box for the mold.<br />
|'''Figure 3-b''' The final mold design for our millifluid PDMS chip used for cell-to-cell communication experiments. <br />
|'''Figure 3-c''' A design for a 96-well plate with connected wells, which allows automated measurements in a plate reader.<br />
|}<br />
<br />
<br />
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.<br />
<br />
<html></article></html><br />
<br />
<html><article></html><br />
<br />
==3D-Printing and Rapid Prototyping==<br />
<br />
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. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 MakerBot.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerWare.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerBot ABS.jpg|x200px]]<br />
|-<br />
|'''Figure 4-a''' The MakerBot Replicator (2nd generation) we used to print our molds.<br />
|'''Figure 4-b''' 'Screenshot' of the MakerWare software we used to print our molds.<br />
|'''Figure 4-c''' A roll of ABS filament used by the 3D-printer.<br />
|}<br />
<br />
<br />
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).<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_comb_and_box.jpg|200px]]<br />
|[[File:ETH Zurich 2014 small grid.JPG|200px]]<br />
|[[File:ETH Zurich diffusion plate.JPG|200px]]<br />
|[[File:ETH Zurich 2014 96 well all connected.jpeg|200px]]<br />
|-<br />
|'''Figure 5-a''' Printed gel-comb and box. The box was autoclaved at 121 ⁰C. <br />
|'''Figure 5-b''' Printed millifluid grid with interconnected wells (edge length of 3 mm).<br />
|'''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).<br />
|'''Figure 5-d''' Printed 96-well plate, all wells (edge length of 5 mm) are connected.<br />
|}<br />
<br />
<html></article></html><br />
<br />
<html><article id='Preparation'></html><br />
<br />
==PDMS Chip Preparation==<br />
<br />
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). The 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]. All the mold shown below were at least used once before the pictures were taken.<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_first_mold_with_PDMS.jpg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion mold.JPG|200px]]<br />
|[[File:ETH Zurich 2014 final mold.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 final mold closeup.jpeg|200px]]<br />
|-<br />
|'''Figure 6-a''' The very first mold design. PDMS stuck between the wells while removing it. <br />
|'''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).<br />
|'''Figure 6-c''' The final mold design for cell-to-cell communication experiments (edge length of 5 mm, channel length of 3 mm). <br />
|'''Figure 6-d''' Close up of the final mold design. The separate layers are clearly visible ('additive' manufacturing).<br />
|}<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 broken chip.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 2 well diffusion chip upsidedown.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 PDMS diffusion chip final.jpeg|200px]]<br />
|[[File:ETH_Zurich_2014_final_chip_zoom.png|200px]]<br />
|-<br />
|'''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.<br />
|'''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).<br />
|'''Figure 7-c''' The final PDMS chip for cell-to-cell communication experiments (edge length of 5 mm, channel length of 3 mm). <br />
|'''Figure 7-d''' Close up of the final PDMS chip. The channels are well defined and even small structures separated evenly from the mold.<br />
|}<br />
<html></article></html><br />
<br />
<html><article id='movies'></html><br />
<br />
==Time-Lapse Movies==<br />
<br />
Below you find an overview of 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 (see video 1). 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. 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.<br />
<br />
<br />
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. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:100%; max-width: 650px; margin: auto;"<br />
|{{: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>}}<br />
|{{: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>}}<br />
|-<br />
|'''Video 1 The very first diffusion experiment with fluorescent highlighter ink (pyranine).''' The wells of the PDMS chip were filled with LB agar. About 5 μL ink were added in a punched whole on one side of the two wells. ~4500x faster than real-time.<br />
|'''Video 2 Diffusion experiment with liquid cultures.''' The wells of the PDMS chip were filled with LB medium, separated by solidified 2% agarose in the channel. The bottom well contained 3OC6-HSL, the top well ''E. coli'' cells with sfGFP under the control of pLux. ~4500x faster than real-time.<br />
|-<br />
|{{: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>}}<br />
|{{: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>}}<br />
|-<br />
|'''Video 3 Diffusion experiment with 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, the top well ''E. coli'' cells with sfGFP under the control of pLux confined in alginate beads (d=3 mm). ~1850x faster than real-time.<br />
|'''Video 4 Cell communication experiment with alginate beads in defined liquid medium.''' The wells of the PDMS chip were filled with CB medium. The bottom well contained ''E. coli'' cells expressing LuxI, which catalyzes the production of 3OC6-HSL; the top well contained ''E. coli'' cells with sfGFP under the control of pLux, All cells were confined in alginate beads (d=3 mm). ~3450x faster than real-time.<br />
|-<br />
|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>}}<br />
|-<br />
|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.<br />
|}<br />
<br />
<br />
<html></article></html><br />
<br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/lab/chipTeam:ETH Zurich/lab/chip2014-10-18T00:42:57Z<p>Mbahls: /* PDMS Chip Preparation */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Millifluidic Chip & Rapid Prototyping}}<br />
<br />
<html><article style='min-height:800px'></html><br />
==Overview==<br />
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).<br />
<br />
[[File:ETH Zurich Rule 6.PNG|300px|thumb|left|'''Figure 1''' '''Classical grid from cellular automata theory''' (ON state=back, OFF state=white).]]<br />
[[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).]]<br />
<br />
<br />
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.<br />
<br><br />
<br><br />
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.<br />
<br />
<html></article></html><br />
<br />
<br />
<html><article></html><br />
<br />
==Mold Design and 3D Print Exchange==<br />
<br />
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>. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_mesh_2_20140826.jpg|200px]]<br />
|[[File:ETH Zurich 2014 final mold model 2.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion plate model.jpeg|200px]]<br />
|-<br />
|'''Figure 3-a''' The first design for a gel-comb, a mold for a millifluidic PDMS chip and a corresponding box for the mold.<br />
|'''Figure 3-b''' The final mold design for our millifluid PDMS chip used for cell-to-cell communication experiments. <br />
|'''Figure 3-c''' A design for a 96-well plate with connected wells, which allows automated measurements in a plate reader.<br />
|}<br />
<br />
<br />
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.<br />
<br />
<html></article></html><br />
<br />
<html><article></html><br />
<br />
==3D-Printing and Rapid Prototyping==<br />
<br />
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. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 MakerBot.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerWare.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerBot ABS.jpg|x200px]]<br />
|-<br />
|'''Figure 4-a''' The MakerBot Replicator (2nd generation) we used to print our molds.<br />
|'''Figure 4-b''' 'Screenshot' of the MakerWare software we used to print our molds.<br />
|'''Figure 4-c''' A roll of ABS filament used by the 3D-printer.<br />
|}<br />
<br />
<br />
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).<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_comb_and_box.jpg|200px]]<br />
|[[File:ETH Zurich 2014 small grid.JPG|200px]]<br />
|[[File:ETH Zurich diffusion plate.JPG|200px]]<br />
|[[File:ETH Zurich 2014 96 well all connected.jpeg|200px]]<br />
|-<br />
|'''Figure 5-a''' Printed gel-comb and box. The box was autoclaved at 121 ⁰C. <br />
|'''Figure 5-b''' Printed millifluid grid with interconnected wells (edge length of 3 mm).<br />
|'''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).<br />
|'''Figure 5-d''' Printed 96-well plate, all wells (edge length of 5 mm) are connected.<br />
|}<br />
<br />
<html></article></html><br />
<br />
<html><article id='Preparation'></html><br />
<br />
==PDMS Chip Preparation==<br />
<br />
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, all other PDMS chips were easily removed without additional aids and placed in clear plastic trays (86 x 128 mm; OmniTrays, Thermo Scientific). The 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]. all the mold shown below were at least used once before the pictures were taken.<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_first_mold_with_PDMS.jpg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion mold.JPG|200px]]<br />
|[[File:ETH Zurich 2014 final mold.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 final mold closeup.jpeg|200px]]<br />
|-<br />
|'''Figure 6-a''' The very first mold design. PDMS stuck between the wells while removing it. <br />
|'''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).<br />
|'''Figure 6-c''' The final mold design for cell-to-cell communication experiments (edge length of 5 mm, channel length of 3 mm). <br />
|'''Figure 6-d''' Close up of the final mold design. The separate layers are clearly visible ('additive' manufacturing).<br />
|}<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 broken chip.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 2 well diffusion chip upsidedown.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 PDMS diffusion chip final.jpeg|200px]]<br />
|[[File:ETH_Zurich_2014_final_chip_zoom.png|200px]]<br />
|-<br />
|'''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.<br />
|'''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).<br />
|'''Figure 7-c''' The final PDMS chip for cell-to-cell communication experiments (edge length of 5 mm, channel length of 3 mm). <br />
|'''Figure 7-d''' Close up of the final PDMS chip. The channels are well defined and even small structures separated evenly from the mold.<br />
|}<br />
<html></article></html><br />
<br />
<html><article id='movies'></html><br />
<br />
==Time-Lapse Movies==<br />
<br />
Below you find an overview of 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 (see video 1). 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. 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.<br />
<br />
<br />
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. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:100%; max-width: 650px; margin: auto;"<br />
|{{: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>}}<br />
|{{: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>}}<br />
|-<br />
|'''Video 1 The very first diffusion experiment with fluorescent highlighter ink (pyranine).''' The wells of the PDMS chip were filled with LB agar. About 5 μL ink were added in a punched whole on one side of the two wells. ~4500x faster than real-time.<br />
|'''Video 2 Diffusion experiment with liquid cultures.''' The wells of the PDMS chip were filled with LB medium, separated by solidified 2% agarose in the channel. The bottom well contained 3OC6-HSL, the top well ''E. coli'' cells with sfGFP under the control of pLux. ~4500x faster than real-time.<br />
|-<br />
|{{: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>}}<br />
|{{: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>}}<br />
|-<br />
|'''Video 3 Diffusion experiment with 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, the top well ''E. coli'' cells with sfGFP under the control of pLux confined in alginate beads (d=3 mm). ~1850x faster than real-time.<br />
|'''Video 4 Cell communication experiment with alginate beads in defined liquid medium.''' The wells of the PDMS chip were filled with CB medium. The bottom well contained ''E. coli'' cells expressing LuxI, which catalyzes the production of 3OC6-HSL; the top well contained ''E. coli'' cells with sfGFP under the control of pLux, All cells were confined in alginate beads (d=3 mm). ~3450x faster than real-time.<br />
|-<br />
|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>}}<br />
|-<br />
|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.<br />
|}<br />
<br />
<br />
<html></article></html><br />
<br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Mbahlshttp://2014.igem.org/Team:ETH_Zurich/lab/chipTeam:ETH Zurich/lab/chip2014-10-18T00:39:39Z<p>Mbahls: /* 3D-Printing and Rapid Prototyping */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Millifluidic Chip & Rapid Prototyping}}<br />
<br />
<html><article style='min-height:800px'></html><br />
==Overview==<br />
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).<br />
<br />
[[File:ETH Zurich Rule 6.PNG|300px|thumb|left|'''Figure 1''' '''Classical grid from cellular automata theory''' (ON state=back, OFF state=white).]]<br />
[[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).]]<br />
<br />
<br />
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.<br />
<br><br />
<br><br />
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.<br />
<br />
<html></article></html><br />
<br />
<br />
<html><article></html><br />
<br />
==Mold Design and 3D Print Exchange==<br />
<br />
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>. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_mesh_2_20140826.jpg|200px]]<br />
|[[File:ETH Zurich 2014 final mold model 2.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion plate model.jpeg|200px]]<br />
|-<br />
|'''Figure 3-a''' The first design for a gel-comb, a mold for a millifluidic PDMS chip and a corresponding box for the mold.<br />
|'''Figure 3-b''' The final mold design for our millifluid PDMS chip used for cell-to-cell communication experiments. <br />
|'''Figure 3-c''' A design for a 96-well plate with connected wells, which allows automated measurements in a plate reader.<br />
|}<br />
<br />
<br />
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.<br />
<br />
<html></article></html><br />
<br />
<html><article></html><br />
<br />
==3D-Printing and Rapid Prototyping==<br />
<br />
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. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 MakerBot.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerWare.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerBot ABS.jpg|x200px]]<br />
|-<br />
|'''Figure 4-a''' The MakerBot Replicator (2nd generation) we used to print our molds.<br />
|'''Figure 4-b''' 'Screenshot' of the MakerWare software we used to print our molds.<br />
|'''Figure 4-c''' A roll of ABS filament used by the 3D-printer.<br />
|}<br />
<br />
<br />
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).<br />
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{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_comb_and_box.jpg|200px]]<br />
|[[File:ETH Zurich 2014 small grid.JPG|200px]]<br />
|[[File:ETH Zurich diffusion plate.JPG|200px]]<br />
|[[File:ETH Zurich 2014 96 well all connected.jpeg|200px]]<br />
|-<br />
|'''Figure 5-a''' Printed gel-comb and box. The box was autoclaved at 121 ⁰C. <br />
|'''Figure 5-b''' Printed millifluid grid with interconnected wells (edge length of 3 mm).<br />
|'''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).<br />
|'''Figure 5-d''' Printed 96-well plate, all wells (edge length of 5 mm) are connected.<br />
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<html><article id='Preparation'></html><br />
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==PDMS Chip Preparation==<br />
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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. 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 [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].<br />
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{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_first_mold_with_PDMS.jpg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion mold.JPG|200px]]<br />
|[[File:ETH Zurich 2014 final mold.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 final mold closeup.jpeg|200px]]<br />
|-<br />
|'''Figure 6-a''' The very first mold design. PDMS stuck between the wells while removing it. <br />
|'''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).<br />
|'''Figure 6-c''' The final mold design for cell-to-cell communication experiments (edge length of 5 mm, channel length of 3 mm). <br />
|'''Figure 6-d''' Close up of the final mold design. The separate layers are clearly visible ('additive' manufacturing).<br />
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{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 broken chip.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 2 well diffusion chip upsidedown.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 PDMS diffusion chip final.jpeg|200px]]<br />
|[[File:ETH_Zurich_2014_final_chip_zoom.png|200px]]<br />
|-<br />
|'''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.<br />
|'''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).<br />
|'''Figure 7-c''' The final PDMS chip for cell-to-cell communication experiments (edge length of 5 mm, channel length of 3 mm). <br />
|'''Figure 7-d''' Close up of the final PDMS chip. The channels are well defined and even small structures separated evenly from the mold.<br />
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==Time-Lapse Movies==<br />
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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 LB agar, holes were punched with a pipette tip and filled with highlighter-ink (pyranine) to visualize diffusion (see video 1). 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. 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.<br />
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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. <br />
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{|class="wikitable" style="background-color: white; text-align:center; width:100%; max-width: 650px; margin: auto;"<br />
|{{: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>}}<br />
|{{: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>}}<br />
|-<br />
|'''Video 1 The very first diffusion experiment with fluorescent highlighter ink (pyranine).''' The wells of the PDMS chip were filled with LB agar. About 5 μL ink were added in a punched whole on one side of the two wells. ~4500x faster than real-time.<br />
|'''Video 2 Diffusion experiment with liquid cultures.''' The wells of the PDMS chip were filled with LB medium, separated by solidified 2% agarose in the channel. The bottom well contained 3OC6-HSL, the top well ''E. coli'' cells with sfGFP under the control of pLux. ~4500x faster than real-time.<br />
|-<br />
|{{: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>}}<br />
|{{: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>}}<br />
|-<br />
|'''Video 3 Diffusion experiment with 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, the top well ''E. coli'' cells with sfGFP under the control of pLux confined in alginate beads (d=3 mm). ~1850x faster than real-time.<br />
|'''Video 4 Cell communication experiment with alginate beads in defined liquid medium.''' The wells of the PDMS chip were filled with CB medium. The bottom well contained ''E. coli'' cells expressing LuxI, which catalyzes the production of 3OC6-HSL; the top well contained ''E. coli'' cells with sfGFP under the control of pLux, All cells were confined in alginate beads (d=3 mm). ~3450x faster than real-time.<br />
|-<br />
|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>}}<br />
|-<br />
|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.<br />
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