Team:Edinburgh/Project

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  <li style="width:13%;"><a href="https://2010.igem.org/Team:Edinburgh/Team" class="dir">team - illuminati</a>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Team/Students">students</a></li>
   <li><a href="https://2010.igem.org/Team:Edinburgh/Team/Students">students</a></li>
   <li><a href="https://2010.igem.org/Team:Edinburgh/Team/Environment">environment</a></li>
   <li><a href="https://2010.igem.org/Team:Edinburgh/Team/Environment">environment</a></li>
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  <li style="width:14%;"><a href="https://2010.igem.org/Team:Edinburgh/Project" class="dir">genomic BRIDGEs</a>
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  <li><a href="https://2010.igem.org/Team:Edinburgh/Project" class="dir">genomic BRIDGEs</a>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Project">the protocol</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Project/Protocol">the protocol</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Project">biobrick parts</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/BioBricks#Genomic">submitted parts</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Project">results</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Results#Genomic">results</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Project">future work</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Project/Future">the future</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Project">references</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Project/References">references</a></li>
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  <li style="width:14%;"><a href="https://2010.igem.org/Team:Edinburgh/Bacterial" class="dir">bacterial BRIDGEs</a>
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  <li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial" class="dir">bacterial BRIDGEs</a>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Core_repressilator">the repressilator</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Core_repressilator">the project</a></li>
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  <li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_producer">red light</a></li>
   <li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_sensor">red sensor</a></li>
   <li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_sensor">red sensor</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Red_light_producer">red producer</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_producer">blue light</a></li>
   <li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Blue_light_sensor">blue sensor</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_producer">green light</a></li>
   <li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Green_light_sensor">green sensor</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/BioBricks#Bacterial">submitted parts</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Results#Bacterial">results</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial">results</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/Future">the future</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial">future work</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Bacterial/References">references</a></li>
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   </ul>
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  <li style="width:15%;"><a href="https://2010.igem.org/Team:Edinburgh/Modelling" class="dir">modelling BRIDGEs</a>
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  <li><a href="https://2010.igem.org/Team:Edinburgh/Modelling" class="dir">modelling BRIDGEs</a>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Modelling">the model</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Kappa">kappa</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Modelling">results</a></li>
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  <li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Genomic">the genomic model</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Modelling">references</a></li>
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  <li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Signalling">the signalling model</a></li>
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  <li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Tools">tools</a></li>
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  <li><a href="https://2010.igem.org/Team:Edinburgh/Results#Modelling">results</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/Future">the future</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Modelling/References">references</a></li>
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  <li style="width:14%;"><a href="https://2010.igem.org/Team:Edinburgh/Human" class="dir">human BRIDGEs</a>
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  <li><a href="https://2010.igem.org/Team:Edinburgh/Human" class="dir">human BRIDGEs</a>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Human">human aspects</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Human/Communication">communication of science</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Human">results</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Human/Branding">iGEM survey</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Human">future work</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Human">references</a></li>
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  <li><a href="https://2010.igem.org/Team:Edinburgh/Human/Epic">the epic</a></li>
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  <li><a href="https://2010.igem.org/Team:Edinburgh/Human/FutureApps">future applications</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Results#Human">further thoughts</a></li>
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  <li><a href="https://2010.igem.org/Team:Edinburgh/Human/References">references</a></li>
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  <li style="width:11%;"><a href="https://2010.igem.org/Team:Edinburgh/Notebook" class="dir">notebooks&nbsp;&nbsp;&nbsp;&nbsp;</a>
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  <li><a href="https://2010.igem.org/Team:Edinburgh/Notebook" class="dir">lab notes&nbsp;&nbsp;&nbsp;</a>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Notebook">collaboration</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Collaboration">collaboration</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Notebook">BRIDGE</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Attribution">attribution</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Notebook">red light</a></li>
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  <li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/BRIDGE">BRIDGE</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Notebook">red sensor</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_producer">red light</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Notebook">blue light</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Red_light_sensor">red sensor</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Notebook">blue sensor</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_producer">blue light</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Notebook">safety</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Blue_light_sensor">blue sensor</a></li>
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   <li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_producer">green light</a></li>
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  <li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Green_light_sensor">green sensor</a></li>
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  <li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Modelling">modelling</a></li>
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  <li><a href="https://2010.igem.org/Team:Edinburgh/Notebook/Safety">safety</a></li>
   <li><a href="http://www.openwetware.org/wiki/French_Lab">protocols</a></li>
   <li><a href="http://www.openwetware.org/wiki/French_Lab">protocols</a></li>
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  <li style="width:8%;"><a href="https://2010.igem.org/Team:Edinburgh/Gallery" class="dir">gallery&nbsp;&nbsp;&nbsp;&nbsp;</a>
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  <li><a href="https://2010.igem.org/Team:Edinburgh/Acknowledgements" class="dir">acknowledgements</a>
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<li style="width:9%;"><a href="https://2010.igem.org/Team:Edinburgh/Acknowledgements" class="dir">acknowledgements</a>
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<a name="Concept" id="Concept"></a><h2>BRIDGE: The concept</h2><br>
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<a name="Introduction" id="Introduction"></a><h2>Genomic BRIDGEs</h2>
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<p>BRIDGE stands for BioBrick Recombination In Direct Genomic Editing. It is a non-standard method of creating BioBricks using homologous recombination instead of restriction digestion to directly insert new genes into the genome, without leaving a marker behind.</p><br>
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<p>Plasmid transformation <b>protocols</b> are the <b>backbone</b> of modern day synthetic biology, allowing for bacteria such as <i>E. coli</i> to take up foreign genetic material and express it as part of their cellular mechanisms. However, since only a relatively low number of cells are actually transformed in the process, selection markers are necessary to <b>identify</b> the cells that have acquired the plasmid; this usually takes the form of an antibiotic resistance gene built into the plasmid, which has the <b>undesired</b> effect of giving the transformed cells resistance to commonly-used antibiotics.</p>
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<center><p><img src="https://static.igem.org/mediawiki/2010/5/5c/Ed10-OriginalBridge.JPG" width="800" height="441" border="0" /></p><br>
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<p>Image: Appl Environ Microbiol. 2008 July; 74(13): 4241–4245 (Fig. 1)</p><br></center>
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<p>What if synthetic biologists were able to <b>utilise</b> an efficient two-step recombination method for markerless gene insertion and deletion? In 2008, Wei Sun, Shifeng Wang, and Roy Curtiss III of Arizona State University <b>published</b> such a protocol, based on the lambda red recombinase system (a simple method for disrupting chromosomal genes in bacteria such as <i>E. coli</i> using PCR products). The 2010 University of Edinburgh iGEM team has <b>adapted</b> their method to take advantage of the reusability of BioBricks, such that synthetic biologists can <b>target</b> critical areas of the <i>E. coli</i> genome with even greater efficiency.</p>
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<p>The first step of BRIDGE requires the deletion of existing DNA (probably a non-coding piece or a non-essential gene) to introduce a construct of two genes; one an antibiotic resistance gene, the other sacB, which prevents the host from growing on sucrose. After the first step we can select for cells which have taken up the construct by growing them on the relevant antibiotic.</p>
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<p><b>BRIDGE</b> stands for BioBrick Recombination In Direct Genomic Editing. It is an alternative <b>method</b> for inserting BioBricks into the genome by using homologous recombination instead of restriction digestion, with the added <b>bonus</b> of not leaving a marker behind in the product.</p><br>
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<p>The second step involves swapping the construct for another piece of DNA (e.g. a BioBrick construct). After this we can select for those with the new gene by growing the cells on sucrose.</p><br>
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<a name="Advantages" id="Advantages"></a><h2>BRIDGE: The advantages</h2><br>
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<p>BRIDGE has a significant advantage over the current method of BioBrick insertion. For one, vector independent - whole PCR constructs can be inserted directly into the genome in two steps in under a week, compared to the lengthy process of vector digestion and ligation required with normal BioBricks.</p>
 
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<p>The other major advantage is that it will not leave a lasting marker in the genome. With most BioBricks we have to leave a marker (antibiotic resistance, GFP, etc) in our constructs so that we can guarantee their presence. This becomes an issue, a) when you want to use the organism in an industrial or environmental capacity, and b) when you want to insert multiple constructs (there is only a limited number of markers out there). With this system, the markers are removed every time you insert a new gene, so they can be used again and again indefinitely. You could essentially replace the entire genome with new genes.</p><br>
 
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<a name="Project" id="Project"></a><h2>Our Project</h2><br>
 
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<p>Our BRIDGE construct will contain chloramphenicol resistance (cat) and sacB. Both it and the desired gene will be inserted by homologous recombination using the lambda red system. For this we will need up and down-stream sequences of genes which we wish to replace.</p>
 
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<p>To prove the principle of BRIDGE we will remove a non-essential, constitutively expressed gene from the <i>E. coli</i> genome and replace it with a well known marker, such as GFP. We also have several genes from a past project idea which we could delete to increase fatty acid synthesis, and further genes we could introduce which will result in the production of long chain alkenes from the excess fatty acids. This is not useful for our current project but it is a nice way to demonstrate the effectiveness of BRIDGE.</p>
 
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<p>Eventually, BRIDGE will be used to introduce whole light producer-sensor constructs (we hope).</p>
 
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<a name="Project" id="Project"></a><h2>Our Project</h2>
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<a name="Addition" id="Addition"></a><h2>Sequential Addition</h2><br>
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<p>One of the future expansions of BRIDGE which we have discussed is using it to directly introduce genes in the genome next to each other without using the BioBrick method before-hand. If you wanted to insert 4 genes with the steps described above, it would take 8 steps. If you do this with the method below it would take 4 steps.</p><br>
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<center><p><img src="https://static.igem.org/mediawiki/2010/7/71/Ed10-SequentialBridge.JPG" width="800" height="549" border="0" /></p><br></center>
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<p>At the first step of the process, the first antibiotic resistance and sacB are introduced alongside the first gene. The antibiotic resistance gene can then be replaced with the next gene and a second antibiotic resistance gene, thereby cycling the antibiotic resistance such that selection is different at each step. At the last step, both markers are removed and the final constructs can be selected for by growth on sucrose (growth on sucrose can also be used as a negative control at each stage).</p>
 
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<p>The final construct would look as below:</p>
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<p>Our BRIDGE construct contains two selection markers in order to <b>successfully</b> complete the protocol (described in greater detail <a href="https://2010.igem.org/Team:Edinburgh/Project/Protocol">here</a>): <i>cat</i>, which confers chloramphenicol resistance, and <i>sacB</i>, which is toxic when the host is grown on sucrose. Both the construct and the desired gene are inserted by homologous recombination using the lambda red recombinase system. For this we <b>require</b> up- and down-stream sequences of the genes that we wish to replace.</p>
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<p>To <b>prove</b> the principle of BRIDGE we removed a non-essential, constitutively expressed gene from the <i>E. coli</i> genome and replaced it with a well-known marker, GFP. This could have been <b>extended</b> further: we also had several genes from a past project idea which we could delete to increase fatty acid synthesis, and other genes we could introduce which would result in the production of long chain alkenes from the excess fatty acids. This is not <b>useful</b> for our current project but it would be a nice way to demonstrate the effectiveness of BRIDGE.</p>
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<p>Eventually, we hoped that BRIDGE could be used to introduce whole light producer-sensor constructs, to <b>demonstrate</b> its ability for utilisation in further work using BioBricks.</p><br>
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<center><p><img src="https://static.igem.org/mediawiki/2010/a/a7/Ed10-FinalBridge.JPG" width="800" height="199" border="0" /></p><br></center>
 
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<a name="Content" id="Content"></a><h2>Table of Contents</h2>
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<a href="https://2010.igem.org/Team:Edinburgh/Project/Protocol">The protocol proper, explaining the technical details of BRIDGE.</a>
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</li>
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<li>
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<a href="https://2010.igem.org/Team:Edinburgh/BioBricks#Genomic">The BioBricks we submitted as part of developing the BRIDGE protocol.</a>
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</li>
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<li>
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<a href="https://2010.igem.org/Team:Edinburgh/Results#Genomic">A summary of what we achieved developing the BRIDGE protocol.</a>
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</li>
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<li>
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<a href="https://2010.igem.org/Team:Edinburgh/Project/Future">Our vision of the future of the BRIDGE protocol, and where we would like to go next.</a>
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</li>
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<li>
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<a href="https://2010.igem.org/Team:Edinburgh/Project/References">References used throughout the section.</a><br>
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<span style="color:ivory;">Throughout this wiki there are words in <b>bold</b> that indicate a relevance to <b>human aspects</b>. It will become obvious that <b>human aspects</b> are a part of almost everything in <b>iGEM</b>.</span>
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Latest revision as of 02:14, 28 October 2010







Genomic BRIDGEs


Plasmid transformation protocols are the backbone of modern day synthetic biology, allowing for bacteria such as E. coli to take up foreign genetic material and express it as part of their cellular mechanisms. However, since only a relatively low number of cells are actually transformed in the process, selection markers are necessary to identify the cells that have acquired the plasmid; this usually takes the form of an antibiotic resistance gene built into the plasmid, which has the undesired effect of giving the transformed cells resistance to commonly-used antibiotics.

What if synthetic biologists were able to utilise an efficient two-step recombination method for markerless gene insertion and deletion? In 2008, Wei Sun, Shifeng Wang, and Roy Curtiss III of Arizona State University published such a protocol, based on the lambda red recombinase system (a simple method for disrupting chromosomal genes in bacteria such as E. coli using PCR products). The 2010 University of Edinburgh iGEM team has adapted their method to take advantage of the reusability of BioBricks, such that synthetic biologists can target critical areas of the E. coli genome with even greater efficiency.

BRIDGE stands for BioBrick Recombination In Direct Genomic Editing. It is an alternative method for inserting BioBricks into the genome by using homologous recombination instead of restriction digestion, with the added bonus of not leaving a marker behind in the product.



Our Project


Our BRIDGE construct contains two selection markers in order to successfully complete the protocol (described in greater detail here): cat, which confers chloramphenicol resistance, and sacB, which is toxic when the host is grown on sucrose. Both the construct and the desired gene are inserted by homologous recombination using the lambda red recombinase system. For this we require up- and down-stream sequences of the genes that we wish to replace.

To prove the principle of BRIDGE we removed a non-essential, constitutively expressed gene from the E. coli genome and replaced it with a well-known marker, GFP. This could have been extended further: we also had several genes from a past project idea which we could delete to increase fatty acid synthesis, and other genes we could introduce which would result in the production of long chain alkenes from the excess fatty acids. This is not useful for our current project but it would be a nice way to demonstrate the effectiveness of BRIDGE.

Eventually, we hoped that BRIDGE could be used to introduce whole light producer-sensor constructs, to demonstrate its ability for utilisation in further work using BioBricks.



Table of Contents





Throughout this wiki there are words in bold that indicate a relevance to human aspects. It will become obvious that human aspects are a part of almost everything in iGEM.