Team:Edinburgh/Project

From 2010.igem.org

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<p>Plasmid transformation protocols are the backbone 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 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.</p>
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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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<p>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 <i>E. coli</i> using PCR products). The 2010 University of Edinburgh iGEM team has adapted their method to take advantage of the reusability of BioBricks, such that biologists can target critical areas of the <i>E. coli</i> genome with even greater efficiency.</p>
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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>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.</p><br>
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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>Our BRIDGE construct contains two selection markers in order to successfully complete the protocol (described in greater detail <a href="http://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 require up- and down-stream sequences of the genes that we wish to replace.</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="http://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 prove 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 extended further: 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 would be a nice way to demonstrate the effectiveness of BRIDGE.</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 demonstrate its ability for utilisation in further work using BioBricks.</p><br>
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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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Revision as of 21:13, 26 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.