Calgary/design

From 2007.igem.org

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<p> Once we had chosed our project the next step was to design a system that would do what we wanted. The steps outlined in this section are the <b>MOST CURRENT </b> used in our project. This section does not describe the primers, plates, and parts that were considered but not used. Or the techniques that were attempted but did not work</p>
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<p> Welcome to our Teams wetalab section. Our wetlab entry consists of several different components, which are described in this section. Just click on the compenent you want to learn more about</p>
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<p style="font-size:18px; font-weight:900"> E. coLisa Project Design </p>
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<p> The following circut was designed to implement our system (image and design schematic pending) </p>
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<p style="font-size:18px; font-weight:900"> E. coLisa Project Preparation Work </p>
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<p> Once a design for the project had been determined our team begin preparing all of the materials and reagents needed to complete our project. The idea was to have everything ready to go for when the biobrick parts arrive.</p>
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<p><a href="#logicCircut" title="See our logic circut" >Logic Circut</a><br />
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<p> Preparing Plates. AMP resistant </p>
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Our logic circut allows for very high control over expression of a desired gene</p>  
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<p> Have everything ready to go for when parts arrive </p>
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<p> Quick list of parts...
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<ul>
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<p>Agarase <br/>
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<li><b> R0084</b> - Light Sensor Promoter </li>
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The protein that degrades agar
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<li><b> R0062</b> - AHL Promoter </li>
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<li><b> R0011</b> - Temperature Sensitive Promotor</li>
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<li><b> S03600</b> - AHL Intermediate</li>
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<li><b> I13544</b> - GFP RBS Terminator</li>
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<li><b> J23008</b> - RNA Key</li>
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<li><b> B0034</b> - RBS</li>
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<li><b> B0015</b> - Terminator</li>
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<li><b> J06501</b> - Temperature Sensitive Component </li>
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<li><b> I51001</b> - ccdb and AMP resistant (death gene used to do plasmid switches</li>
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</ul>
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<p> Wells from registery </p>
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<p> I) Rehydrate the wells </p>
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<p>
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<p>II) 2 micro litres of rehydrated part --> transformed into TOP10 </p>
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BioMarkers <br />
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Ever wanted to draw on bacteria?
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</p>
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<p>
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Light Sensor <br />
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The system that allows are bacteria to see</p>
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</td>
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</tr>
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<hr />
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    <tr>
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      <td style="width:85%;"><p style="font-size:20px; font-weight:bold; text-decoration:underline"><a style="text-decoration:none" name="logicCircut"> Logic Circut</a></p></td>
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      <td width="15%"><a style="float:right;" href="#top" title="return to top">return to top</a> </td>
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<p>
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The sucess of our project is dependent on us having very percise control over the expression of our reporter gene. In order to draw a legible picture our system needs to be able to induce very high expression of agarase under the desired conditions and then almost completely stop agarase expression when the activating conditions are removed. This is because an over expression of agarase might just degarde the entire plate leaving nothing visible as a picture. Initially one of our team members, Dave Curran, designed a very slick and complex system to regulate expression. However as we were running out of time we were forced to rely on a more simplified version. The simplified version works as follows.
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</p>
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<p>
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In the absence of 660nm light and AHL, both promoters in the circuit are repressed.  If the bacteria were then to be exposed to light, the protein LuxR would be produced, but would not activate the promoter lux pR.  When AHL is added to the cells that are still in the dark, the lux pR promoter would still not be activated, as the protein LuxR would not be present.  But if AHL is added to the cells, and they are then exposed to light, both AHL and LuxR will be present, and so the lux pR promoter will be activated.  When this occurs the reporter gene is expressed, and more LuxR is produced so that the system will remain on even when the light is then taken away.</p>
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<p>
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The complicated system functions in much the same way, except it contains one additional level of security, to prevent accidental activation of the system.  There is an RNA lock covering the ribosome binding site in front of the second luxR in the plasmid.  This way, even if the lux pR promoter is a touch leaky, no LuxR will be produced to fully activate the system. 
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</p>
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<p>
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The application below shows the schematics of both the complex and simple systems. Hovering over a part with the mouse will highlight its corresponding description in the table. Clicking on a part in the diagram will open the registry's page that desribes the part.
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</p>
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Revision as of 00:16, 26 October 2007

back to U of C Homepage Check out evoGEM
Projects Design: Wet Lab Design: Printer Design: Software Testing Construction: The Wetlab Protocols Final Result of E.co Lisa

Welcome to our Teams wetalab section. Our wetlab entry consists of several different components, which are described in this section. Just click on the compenent you want to learn more about

Logic Circut
Our logic circut allows for very high control over expression of a desired gene

Agarase
The protein that degrades agar

BioMarkers
Ever wanted to draw on bacteria?

Light Sensor
The system that allows are bacteria to see

Logic Circut

 return to top

The sucess of our project is dependent on us having very percise control over the expression of our reporter gene. In order to draw a legible picture our system needs to be able to induce very high expression of agarase under the desired conditions and then almost completely stop agarase expression when the activating conditions are removed. This is because an over expression of agarase might just degarde the entire plate leaving nothing visible as a picture. Initially one of our team members, Dave Curran, designed a very slick and complex system to regulate expression. However as we were running out of time we were forced to rely on a more simplified version. The simplified version works as follows.

In the absence of 660nm light and AHL, both promoters in the circuit are repressed. If the bacteria were then to be exposed to light, the protein LuxR would be produced, but would not activate the promoter lux pR. When AHL is added to the cells that are still in the dark, the lux pR promoter would still not be activated, as the protein LuxR would not be present. But if AHL is added to the cells, and they are then exposed to light, both AHL and LuxR will be present, and so the lux pR promoter will be activated. When this occurs the reporter gene is expressed, and more LuxR is produced so that the system will remain on even when the light is then taken away.

The complicated system functions in much the same way, except it contains one additional level of security, to prevent accidental activation of the system. There is an RNA lock covering the ribosome binding site in front of the second luxR in the plasmid. This way, even if the lux pR promoter is a touch leaky, no LuxR will be produced to fully activate the system.

The application below shows the schematics of both the complex and simple systems. Hovering over a part with the mouse will highlight its corresponding description in the table. Clicking on a part in the diagram will open the registry's page that desribes the part.

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