Team:ETH Zurich/Infoproc

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<h1>Information processing</h1>
<h1>Information processing</h1>
<p align="justify">We have two bacterial strains: Firstly the mine strain which provides the sender cells and secondly the non-mine strain which provides the receiver cells. <br><br> <b> Signal origin</b>: The sender colony secretes the signaling molecule 3-oxo-N-hexanoyl-L-homoserine lactone  
<p align="justify">We have two bacterial strains: Firstly the mine strain which provides the sender cells and secondly the non-mine strain which provides the receiver cells. <br><br> <b> Signal origin</b>: The sender colony secretes the signaling molecule 3-oxo-N-hexanoyl-L-homoserine lactone  
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([https://2013.igem.org/Team:ETH_Zurich/Experiments_2 AHL])by constitutive [https://2013.igem.org/Team:ETH_Zurich/pre_proc LuxI activation]. <br><br> <b>Pre-Processing</b> : The agar minefield consists of colonies in a hexagonal grid with mine and non-mine colonies. The secreted AHL [https://2013.igem.org/Team:ETH_Zurich/Experiments_2#diffusion_experiment diffuses] through  the agar. This leads to differnet possible receiver situations: 0 mines, 1 mine and 2 mines around (to know more about the reasons for this set-up please click [https://2013.igem.org/Team:ETH_Zurich/pre_proc here]).In the receiver cells, the signaling molecule forms a complex with the inactive LuxR to activate it.<br><br><b>Processing</b>: By using [https://2013.igem.org/Team:ETH_Zurich/Processing_2 different sensitive P<sub>LuxR</sub> promoters] the AHL input concentration is translated into different outputs using specific reporters : GFP/RFP and later different
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([https://2013.igem.org/Team:ETH_Zurich/Experiments_2 AHL])by constitutive [https://2013.igem.org/Team:ETH_Zurich/pre_proc LuxI activation]. <br><br> <b>Pre-Processing</b> : the agar minefield consists of colonies in a hexagonal grid with mine and non-mine colonies. The secreted AHL [https://2013.igem.org/Team:ETH_Zurich/Experiments_2#diffusion_experiment diffuses] through  the agar. This leads to differnet possible receiver situations: 0 mines, 1 mine and 2 mines around (to know more about the reasons for this set-up please click [https://2013.igem.org/Team:ETH_Zurich/pre_proc here]).In the receiver cells, the signaling molecule forms a complex with the inactive LuxR to activate it.<br><br><b>Processing</b>: by using [https://2013.igem.org/Team:ETH_Zurich/Processing_2 different sensitive P<sub>LuxR</sub> promoters] the AHL input concentration is translated into different outputs using specific reporters : GFP/RFP and later different
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[https://2013.igem.org/Team:ETH_Zurich/Experiments_7 hydrolases].<br><br><b>Optimization</b>: After the proof-of-principle with GFP we carried out the first trials using the hydrolases as reporter system. We then concluded that our reporter system set-up is leaky and start to review the circuit to reduce the leakiness. (Please see the optimization part for more [https://2013.igem.org/Team:ETH_Zurich/Circuit details]).<br><br><b> Player interaction </b>: [https://2013.igem.org/Team:ETH_Zurich/Play the player] pipets a substrate-mix on a colony, which leads to a color change of the colony. This gives the player information to logically carry out the next move in the game. So the left click in the computer game is pipetting a substrate mixture on the colony in the bio-game. As a goodie we also included the right click in the computer game (flagging) by adding either a flagging solution converted by the chromosomal expressed lacZ or by adding the <i>Remazol blue dye</i> which is, in contrats to the lacZ flagging, removable.<br><br><b>Output</b>: Within <b>minutes</b> after the addition of the substrate-mix a change in color due to the conversion of the specific substrates by the different expressed hydrolases according to the different amount of AHL dependent on the number of surrounding mines, indicates the identity of the played colony and number of surrounding  mine colonies.</p> <br clear="all">[[File:Infoproc24.png|1250px|center|thumb|<b>Figure 1:Information processing from secreted signaling molecule to colorimetric response.</b> The signal diffuses through the agar from sender cells (light blue) to receiver cells (dark blue).The non-mine colonies are designed to distinguish between different concentrations of AHL and translate this information into expression of different hydrolases. The expression is driven by different P<sub>LuxR</sub> promoters that show different AHL sensitivities and serve as high pass filters. After an incubation time of 12 hours the player  pipets a substrate on the colony. The hydrolase converts the substrate into a colored product which is visible by eye ]]
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[https://2013.igem.org/Team:ETH_Zurich/Experiments_7 hydrolases].<br><br><b>Optimization</b>: after the proof-of-principle with GFP we carried out the first trials using the hydrolases as reporter system. We then concluded that our reporter system set-up is leaky and start to review the circuit to reduce the leakiness. (Please see the optimization part for more [https://2013.igem.org/Team:ETH_Zurich/Circuit details]).<br><br><b> Player interaction </b>: [https://2013.igem.org/Team:ETH_Zurich/Play the player] pipets a substrate-mix on a colony, which leads to a color change of the colony. This gives the player information to logically carry out the next move in the game. So the left click in the computer game is pipetting a substrate mixture on the colony in the bio-game. As a goodie we also included the right click in the computer game (flagging) by adding either a flagging solution converted by the chromosomal expressed lacZ or by adding the <i>Remazol blue dye</i> which is, in contrats to the lacZ flagging, removable.<br><br><b>Output</b>: within <b>minutes</b> after the addition of the substrate-mix a change in color due to the conversion of the specific substrates by the different expressed hydrolases according to the different amount of AHL dependent on the number of surrounding mines, indicates the identity of the played colony and number of surrounding  mine colonies.</p> <br clear="all">[[File:Infoproc24.png|1250px|center|thumb|<b>Figure 1:Information processing from secreted signaling molecule to colorimetric response.</b> The signal diffuses through the agar from sender cells (light blue) to receiver cells (dark blue).The non-mine colonies are designed to distinguish between different concentrations of AHL and translate this information into expression of different hydrolases. The expression is driven by different P<sub>LuxR</sub> promoters that show different AHL sensitivities and serve as high pass filters. After an incubation time of 12 hours the player  pipets a substrate on the colony. The hydrolase converts the substrate into a colored product which is visible by eye ]]
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Revision as of 10:12, 25 October 2013

Header2.png
80px-Eth igem logo.png

Information processing

We have two bacterial strains: Firstly the mine strain which provides the sender cells and secondly the non-mine strain which provides the receiver cells.

Signal origin: The sender colony secretes the signaling molecule 3-oxo-N-hexanoyl-L-homoserine lactone (AHL)by constitutive LuxI activation.

Pre-Processing : the agar minefield consists of colonies in a hexagonal grid with mine and non-mine colonies. The secreted AHL diffuses through the agar. This leads to differnet possible receiver situations: 0 mines, 1 mine and 2 mines around (to know more about the reasons for this set-up please click here).In the receiver cells, the signaling molecule forms a complex with the inactive LuxR to activate it.

Processing: by using different sensitive PLuxR promoters the AHL input concentration is translated into different outputs using specific reporters : GFP/RFP and later different hydrolases.

Optimization: after the proof-of-principle with GFP we carried out the first trials using the hydrolases as reporter system. We then concluded that our reporter system set-up is leaky and start to review the circuit to reduce the leakiness. (Please see the optimization part for more details).

Player interaction : the player pipets a substrate-mix on a colony, which leads to a color change of the colony. This gives the player information to logically carry out the next move in the game. So the left click in the computer game is pipetting a substrate mixture on the colony in the bio-game. As a goodie we also included the right click in the computer game (flagging) by adding either a flagging solution converted by the chromosomal expressed lacZ or by adding the Remazol blue dye which is, in contrats to the lacZ flagging, removable.

Output: within minutes after the addition of the substrate-mix a change in color due to the conversion of the specific substrates by the different expressed hydrolases according to the different amount of AHL dependent on the number of surrounding mines, indicates the identity of the played colony and number of surrounding mine colonies.


Figure 1:Information processing from secreted signaling molecule to colorimetric response. The signal diffuses through the agar from sender cells (light blue) to receiver cells (dark blue).The non-mine colonies are designed to distinguish between different concentrations of AHL and translate this information into expression of different hydrolases. The expression is driven by different PLuxR promoters that show different AHL sensitivities and serve as high pass filters. After an incubation time of 12 hours the player pipets a substrate on the colony. The hydrolase converts the substrate into a colored product which is visible by eye




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