Team:KAIST Korea

From 2013.igem.org

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<div id="top-img-description-box"><span id="top-img-description">Home : Introduction</span></div>
 
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        <h1> Abstract</h1>
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<span id="sub-title">Reguli</span></br></br>
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<span id="little"></span><span id="starter">B</span>iological engineering has brought us handy tools to produce desired products. However, we have to induce genes to properly express them on correct cell phase. We, KAIST 2012 iGEM team, suggest a module that automatically turns the transcription of gene on or off using dual-phase switching module. In our model, promoter orientation is designed to be reversed when sufficient quorum molecule is detected. Then, genes on the other side, indigo synthesizing enzyme bFMO, lactonase, and excisionase are expressed. Indigo is meaningful in that it is the end-product of its metabolic pathway and, its color can be easily detected. Lactonase quenches quorum molecule(lactone), which will generate an oscillating pattern. Excisionase acts on the recombination site to set promoter to its initial orientation.  In this way, we can regulate metabolic pathways. So we call it regulative E.coli, Reguli.
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<li><a style="font-size:14px;" href="https://2012.igem.org/Team:KAIST_Korea/Project_Background">Overview</a></li>
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<li><a style="font-size:14px;" href="https://2012.igem.org/Team:KAIST_Korea/Project_Future">Future Plan</a></li>
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<ul style="width:100px;height:19px;">
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<li><a style="font-size:14px;" href="https://2012.igem.org/Team:KAIST_Korea/Project_Modeling">Modeling</a></li>
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<li><a style="font-size:14px;" href="https://2012.igem.org/Team:KAIST_Korea/Plus_Safety">Safety</a></li>
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<li><a style="font-size:14px;" href="https://2012.igem.org/Team:KAIST_Korea/Plus_Human">Human Practice</a></li>
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<li><a style="font-size:14px;" href="https://2012.igem.org/Team:KAIST_Korea/Plus_Attribution">Attribution</a></li>
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<li><a style="font-size:14px;" href="https://2012.igem.org/Team:KAIST_Korea/Plus_Sponsor">Sponsors</a></li>
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<div id="contact-us-title">2013 KAIST Korea</div>
 +
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<b>Mail</b> : <a href="mailto:kaist.igem.2012@gmail.com">kaist.igem.2012@gmail.com</a></br>
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        <h1> Introduction</h1>
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<span id="sub-title">"We suggest an auto-regulation module free from induction which utilizes dual-phase switching system."</span></br></br>
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<!-- <ul>
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<span id="little"></span><span id="starter">T</span>hroughout past iGEM competitions, many kinds of bio-modules were proposed and tested. They were great and some were brilliant, but it doesn’t seem many of them are universally available. Of course the goal of iGEM is to enrich the database, but we view the beauty of this registry is to contain as many modules which can be readily applied to actual research as possible. Thus, here now, we suggest an auto-regulation module free from induction which utilizes dual-phase switching system and quorum sensing.
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<span id="little2"></span>We may think of many meanings for this module; bio-computing, in-cell signal processing, and auto-control of cell metabolism. For example, since we use dual-phase switching system, we may consider each direction of gene expression as a signal; signal 0 or 1 of binary code. Thereby we can use this module to generate in vivo logic gates or computational system.
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</br></br>
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<img id="top-img" src="https://static.igem.org/mediawiki/2012/6/6d/Back_home.PNG"/>
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<span id="little2"></span>Dual-phase switching system adopts DNA recombination system of bacteriophage origin. In this system, DNA integrase recognizes specific sequences called attB and attP and then invert the sequence between them. Exisionase revert this sequence into its original state by recognizing the recombination site. We can thereby use this system to turn the pathway on or off.
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                </br></br>
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<span id="little2"></span>Products of LuxI and LuxR are used to generate signals in our quorum sensing model. They initiate the operation of auto-regulation module. Lactonase will regulate the signal to yield an oscillating pattern.
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<span id="little2"></span>As our end product, we selected bio-indigo which comes from indole. This widespread pigment is produced by bFMO, bacterial Flavin-containing MonoOxygenase. Successful production of bio-indigo verifies that our module can be embodied well in metabolic pathway and also proposes its potential usage.
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<h1>Project : Overview</h1>
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<span id="little"><span id="starter">T</span>he widely used gene knock-out strategies are based upon a mere [on-off] control and more complex regulation is impossible. On the other hand, knock-down strategies are more versatile in usage but the current techniques are vastly laborious for systematic control and pose difficulty in multiple targeting because of the intricate design and scar accumulation.
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iGEM KAIST 2013 seeks to take the yet-to-be-perfected genetic regulation technology to a new level: The <span id="starter">RAPTOR</span> (RNA Associated Prokaryotic Transcript Optimization with CRISPR) technology. RAPTOR uses CRISPR type III system for gene regulation. CRISPR type III targets and cleaves specific mRNA sequences through complementary binding of crRNA and nuclease activity of Cmr protein complex. RAPTOR exploits this nature of CRISPR for systematically down regulating the mRNA level of specific genes.
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Due to the characteristics of RAPTOR, one of the most useful applications of this technology can be biosynthesis pathway regulation. Programmed crRNA expression allows fine-tuning of target mRNA expression levels and can precisely regulate enzyme concentrations. Not only fine-tuning but multiplex engineering is also possible through simultaneous expression of different crRNAs. Thus, multiple enzymes can be targeted in pathway control.
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As a proof of concept we sought to regulate the lycopene pathway that is incorporated into the genome of Escherichia coli. The efflux of lycopene precursors is optimally reduced through lowering the mRNA levels of enzymes that synthesize non-lycopene end products. Through observation of increased lycopene yield in the E.coli, potentiality of RAPTOR technology can be validated.
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Latest revision as of 10:12, 23 August 2013

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Project : Overview



The widely used gene knock-out strategies are based upon a mere [on-off] control and more complex regulation is impossible. On the other hand, knock-down strategies are more versatile in usage but the current techniques are vastly laborious for systematic control and pose difficulty in multiple targeting because of the intricate design and scar accumulation. iGEM KAIST 2013 seeks to take the yet-to-be-perfected genetic regulation technology to a new level: The RAPTOR (RNA Associated Prokaryotic Transcript Optimization with CRISPR) technology. RAPTOR uses CRISPR type III system for gene regulation. CRISPR type III targets and cleaves specific mRNA sequences through complementary binding of crRNA and nuclease activity of Cmr protein complex. RAPTOR exploits this nature of CRISPR for systematically down regulating the mRNA level of specific genes. Due to the characteristics of RAPTOR, one of the most useful applications of this technology can be biosynthesis pathway regulation. Programmed crRNA expression allows fine-tuning of target mRNA expression levels and can precisely regulate enzyme concentrations. Not only fine-tuning but multiplex engineering is also possible through simultaneous expression of different crRNAs. Thus, multiple enzymes can be targeted in pathway control. As a proof of concept we sought to regulate the lycopene pathway that is incorporated into the genome of Escherichia coli. The efflux of lycopene precursors is optimally reduced through lowering the mRNA levels of enzymes that synthesize non-lycopene end products. Through observation of increased lycopene yield in the E.coli, potentiality of RAPTOR technology can be validated.




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