Team:Calgary/Project/BsDetector/GeneticCircuit

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<h1>Genetic Circuit</h1>
<h1>Genetic Circuit</h1>
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<p>Our pathogen detection system consists of two collections of interconnected genes (operons) placed inside specific regions (loci) of the chromosome. The first operon is designated the "reporter" and consists of a constitutive promoter (<i>P<sub>veg</sub></i>), a ribosomal binding site, a repressible operator (C2P22), and (<i>lacZ</i>), a gene capable of producing a blue pigment (5,5'-dibromo-4,4'-dichloro-indigo) in the presence of X-gal. The blue pigment is made when the enzyme beta-galactosidase, encoded by <i>lacZ</i>, cleaves the X-gal molecule. Our "reporter" operon works in tandem with our "repressor" operon, which consists of the same promoter, ribosomal binding site, and a repressor gene designed to negate the function of the aforementioned operator gene (C2P22). Repression of the operator within the reporter operon is made possible by the unique repressor proteins produced by the repressor gene. The repressor gene is flanked by sequences homologous to a conserved target sequence of our intended pathogen of interest, such that we can identify any strain present through homologous recombination. </p>
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<p>Our pathogen detection system consists of two interacting genes located within the genome of <i>B. subtilis</i>. The reporter gene consists of a constitutive promoter (<i>P<sub>veg</sub></i>), a repressible promoter, a ribosomal binding site (RBS), and a reporter coding sequence. Our preferred reporter of choice is <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1444017"><i>lacZ</i></a>. The <i>lacZ</i> gene corresponds to beta-galactosidase, an enzyme catalyzing the breakdown of X-gal to produce indigo (5,5'-dibromo-4,4'-dichloro-indigo), a dark blue pigment. We decided upon an enzymatic reporter instead of a chromophoric protein reporter. The signal of produced by a chromophoric protein is dependent upon, and proportional to the concentration of protein produced; an <span class="Blue">enzymatic reporter</span> is able to continuously produce a signal that is relatively independent of enzyme concentration allowing a greater signal intensity.</p>
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<p>By default, when the two operons are placed inside <i>B. subtilis</i>, the repressor gene will act upon the operator of our reporter and prevent the transcription of <i>lacZ</i>). With the chromophore not being produced, the <i>B. subtilis</i> will refrain from exhibiting a colorimetric output and will remain in the "negative state". However, when a particular pathogen is introduced to our engineered <i>B. subtilis</i>, it will uptake the target sequence contained within the pathogen through homologous recombination. Essentially, the repressor gene, which is flanked by DNA regions homologous to the target sequence, will be replaced by the target sequence and "knocked off" the chromosome. With the repressor gene absent, the reporter operon will be at liberty to produce the chromophore and cause <i>B. subtilis</i> to yield a blue colorimetric output.</p>
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<p>Our genetic circuit consists of a reporter gene regulated by the repressor gene. In the default state, expression of the reporter is inhibited by the repressor bound to the repressible promoter upstream of the reporter. Activation occurs by homologous recombination between the repressor gene and the target DNA sequence. By flanking the repressor gene with sequences homologous to the target DNA, we are able to knock-out the repressor gene and thus allowing expression of our reporter.</p>
<img src="https://static.igem.org/mediawiki/2014/d/d8/Ucalgary2014genediagram.png" width="500px" class="Center">
<img src="https://static.igem.org/mediawiki/2014/d/d8/Ucalgary2014genediagram.png" width="500px" class="Center">
<p><center><b>Figure 1:</b> DNA detection via homologous recombination.
<p><center><b>Figure 1:</b> DNA detection via homologous recombination.
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<i><b>A)</b></i> Reporter operon consisting of a constitutive promoter (grey arrow), ribosome-binding site (RBS; semicircle), operator (orange), and coding sequence of a reporter (<i>lacZ</i>). Expression of the reporter is repressed in the default state.
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<i><b>A)</b></i> Reporter gene consisting of a constitutive promoter (grey arrow), ribosome-binding site (RBS; semicircle), operator (orange), and coding sequence of a reporter (<i>lacZ</i>). Expression of the reporter is repressed in the default state.
<i><b>B)</b></i> Repressor system for regulation of reporter expression.
<i><b>B)</b></i> Repressor system for regulation of reporter expression.
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<i><b>i)</b></i> Repressor operon consisting of a repressor driven by a constitutive promoter. Expression of the repressor inhibits the expression of the reporter - the repressor specifically targets the operator shown in <i><b>a)</b></i>. Blue regions indicate flanking sequences that are homologous to the amplified target DNA as shown in <i><b>ii)</b></i>. Upon homologous recombination (dotted lines), the disruption of the repressor operon leads to active reporter expression.  
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<i><b>i)</b></i> Repressor gene consisting of a repressor driven by a constitutive promoter. Expression of the repressor inhibits the expression of the reporter - the repressor specifically targets the operator shown in <i><b>a)</b></i>. Blue regions indicate flanking sequences that are homologous to the amplified target DNA as shown in <i><b>ii)</b></i>. Upon homologous recombination (dotted lines), the disruption of the repressor gene leads to active reporter expression.  
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<i><b>ii)</b></i> Target DNA from pathogen, amplified via isothermal-PCR (iPCR). Blue regions indicate sequences homologous to the flanking regions of the repressor operon as shown in <i><b>i)</b></i>.</p></center>
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<i><b>ii)</b></i> Target DNA from pathogen, amplified via isothermal-PCR (iPCR). Blue regions indicate sequences homologous to the flanking regions of the repressor gene as shown in <i><b>i)</b></i>.</p></center>
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<p>A major strength of our project design lies in its high level of customization and modularity. Using our reporter and repressor operons, we can in theory facilitate the detection of any pathogen that might be present within a blood sample simply by modifying the homologous flanking regions of our repressor gene with the proper sequences. To make this possible, we would only need to identify beforehand a target sequence for our pathogen of interest by using a public genome database . Our experiments have shown that a mere 150bp target sequence is sufficient for homologous recombination. In theory, our device should be able to detect any pathogen whose genome has been sequenced to an adequate degree and has been made publicly available.
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<p>A major strength of our project design lies in its high level of customization and modularity. Using our reporter and repressor genes, we can in theory facilitate the detection of any pathogen that might be present within a blood sample simply by modifying the homologous flanking regions of our repressor gene with the proper sequences. To make this possible, we would only need to identify beforehand a target sequence for our pathogen of interest by using a public genome database . Our experiments have shown that a mere 150bp target sequence is sufficient for homologous recombination. In theory, our device should be able to detect any pathogen whose genome has been sequenced to an adequate degree and has been made publicly available.
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<p>In keeping with the consistent record of safety present in iGEM, we have also taken cautious measures to ensure that our engineered <i>B. subtilis</i> cells are virtually harmless in the unlikely event that they escape the casing of our device. The reporter and repressor genes have been placed into the <i>thrC</i> locus of <i>B. subtilis</i> and have effectively replaced the genes that were originally present in this location. Because the <i>thrC</i> gene is needed for the synthesis of threonine - an essential amino acid - replacing it with our operons makes <i>B. subtilis</i> incapable of producing endogenous threonine and thus renders it auxotrophic. Any amount of <i>B. subtilis</i> which may escape our device would be unable to replicate and die promptly.</p>
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<p>In keeping with the consistent record of safety present in iGEM, we have also taken cautious measures to ensure that our engineered <i>B. subtilis</i> cells are virtually harmless in the unlikely event that they escape the casing of our device. The reporter and repressor genes have been placed into the <i>thrC</i> locus of <i>B. subtilis</i> and have therefore replaced the genes that were originally present at that locus. Because the <i>thrC</i> gene is needed for the synthesis of threonine - an essential amino acid - replacing it with our genes makes <i>B. subtilis</i> incapable of producing endogenous threonine and thus renders it auxotrophic. Any amount of <i>B. subtilis</i> which may escape our device would be unable to replicate and die promptly.</p>

Latest revision as of 03:59, 18 October 2014

Genetic Circuit

Our pathogen detection system consists of two interacting genes located within the genome of B. subtilis. The reporter gene consists of a constitutive promoter (Pveg), a repressible promoter, a ribosomal binding site (RBS), and a reporter coding sequence. Our preferred reporter of choice is lacZ. The lacZ gene corresponds to beta-galactosidase, an enzyme catalyzing the breakdown of X-gal to produce indigo (5,5'-dibromo-4,4'-dichloro-indigo), a dark blue pigment. We decided upon an enzymatic reporter instead of a chromophoric protein reporter. The signal of produced by a chromophoric protein is dependent upon, and proportional to the concentration of protein produced; an enzymatic reporter is able to continuously produce a signal that is relatively independent of enzyme concentration allowing a greater signal intensity.

Our genetic circuit consists of a reporter gene regulated by the repressor gene. In the default state, expression of the reporter is inhibited by the repressor bound to the repressible promoter upstream of the reporter. Activation occurs by homologous recombination between the repressor gene and the target DNA sequence. By flanking the repressor gene with sequences homologous to the target DNA, we are able to knock-out the repressor gene and thus allowing expression of our reporter.

Figure 1: DNA detection via homologous recombination. A) Reporter gene consisting of a constitutive promoter (grey arrow), ribosome-binding site (RBS; semicircle), operator (orange), and coding sequence of a reporter (lacZ). Expression of the reporter is repressed in the default state. B) Repressor system for regulation of reporter expression. i) Repressor gene consisting of a repressor driven by a constitutive promoter. Expression of the repressor inhibits the expression of the reporter - the repressor specifically targets the operator shown in a). Blue regions indicate flanking sequences that are homologous to the amplified target DNA as shown in ii). Upon homologous recombination (dotted lines), the disruption of the repressor gene leads to active reporter expression. ii) Target DNA from pathogen, amplified via isothermal-PCR (iPCR). Blue regions indicate sequences homologous to the flanking regions of the repressor gene as shown in i).

A major strength of our project design lies in its high level of customization and modularity. Using our reporter and repressor genes, we can in theory facilitate the detection of any pathogen that might be present within a blood sample simply by modifying the homologous flanking regions of our repressor gene with the proper sequences. To make this possible, we would only need to identify beforehand a target sequence for our pathogen of interest by using a public genome database . Our experiments have shown that a mere 150bp target sequence is sufficient for homologous recombination. In theory, our device should be able to detect any pathogen whose genome has been sequenced to an adequate degree and has been made publicly available.

In keeping with the consistent record of safety present in iGEM, we have also taken cautious measures to ensure that our engineered B. subtilis cells are virtually harmless in the unlikely event that they escape the casing of our device. The reporter and repressor genes have been placed into the thrC locus of B. subtilis and have therefore replaced the genes that were originally present at that locus. Because the thrC gene is needed for the synthesis of threonine - an essential amino acid - replacing it with our genes makes B. subtilis incapable of producing endogenous threonine and thus renders it auxotrophic. Any amount of B. subtilis which may escape our device would be unable to replicate and die promptly.

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