Difference between revisions of "Team:Manchester/Description"

 
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<h1>Project Overview </h1>
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<h1 class="title11">Project Overview </h1>
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   <h1 style="font-size:20px"> Mechanism 1</h1>
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   <h1 class="mechanismm"> Mechanism 1</h1>
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   <h1 style="font-size:40px; color:orange; text-shadow: 1px 1px yellow">Cell Free Mechanism</h1>
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   <h1 class="mectitle">Cell Free System</h1>
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  <img style="width:60%;margin:auto" src="https://static.igem.org/mediawiki/2016/8/86/T--Manchester--mechanism1_overview.png" alt="Mechanism 2 overview diagram" />
 
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<br /><br /><br /><br /><br /><br /><br /><br /><br />
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  <h1 style="font-size:20px"> Mechanism 2</h1>
 
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  <h1 style="font-size:40px; color:orange; text-shadow: 1px 1px yellow">Inducible Gene Switch</h1>
 
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   <p style="font-size: 20px;text-align:left">The <i>alc</i> gene expression system is one of the most reliable chemically inducible gene switches for use in plants and fungus.   This system relies on the ability of AlcR, a transcription factor, to bind to its target <i>alcA</i> promoter (alcA<sup>P</sup>). Based on this, we have engineered  <i>Escherichia coli</i> K-12 derivative DH5α and BL21 to induce expression of chromoproteins when AlcR binds to alcA<sup>P</sup> in the presence of ethanol <sup>[1]</sup>.  
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   <p style="font-size:18px;text-align:left">Enzymatic colourimetric assays are used to determine the concentration of a chemical in a solution by the conversion of a chromogen substrate into a coloured product. We have introduced a plasmid expressing recombinant Alcohol Oxidase 1 (<a href="http://parts.igem.org/Part:BBa_K2092000" target="_blank">AOx</a>) from <i>Pichia pastoris</i> into <i>Escherichia coli</i>  BL21 (DE3) strain that will then be used in the cell-free colorimetric system. This method involves the usage of AOx to oxidise ethanol, producing hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) as a by-product. H<sub>2</sub>O<sub>2</sub> is used as an oxidising agent by horseradish peroxidase (HRP) to convert ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) to produce the colour change <sup>[1]</sup>.
 
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   <div class="directlink"><h1><a href="https://2016.igem.org/Team:Manchester/Description/mechanism2">Click here for more info ></a></h1></div>
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   <div class="directlink"><a href="https://2016.igem.org/Team:Manchester/Description/mechanism1"><h1>Click here for more info </h1></a></div>
 
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  <h1 class="mechanismm1" > Mechanism 2</h1>
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  <h1 class="mectitle1">Inducible Gene Switch</h1>
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  <br /><br />
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<div class="column twothird_size">
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  <img class="width90" style="margin:auto" src="https://static.igem.org/mediawiki/2016/1/12/T--Manchester--mech2_overview.png" alt="Mechanism 2 overview diagram" />
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<div class="info2">
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  <p style="font-size: 18px;text-align:left">The <i>alc</i> gene expression system is one of the most reliable chemically inducible gene switches for use in plants <sup>[2]</sup> and fungus <sup>[3]</sup>.
 +
  This system relies on the ability of <a href="http://parts.igem.org/Part:BBa_K2092001" target="_blank"> AlcR</a>, an alcohol-activated transcription factor, to bind to its target <i>alcA</i> promoter (<a href="http://parts.igem.org/Part:BBa_K2092002" target="_blank">P<i>alc</i>A</a>). Based on this, we have engineered  <i>E. coli</i> K-12 derivative DH5α and BL21 to induce expression of chromoproteins when AlcR binds to the native P<i>alc</i>A and variant of P<i>alc</i>A (<a href="http://parts.igem.org/Part:BBa_K2092003" target="_blank"> P<i>alc</i>A(var)</a>) in the presence of ethanol <sup>[4]</sup>.
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    <br /><br />
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  <div class="directlink"><a href="https://2016.igem.org/Team:Manchester/Description/mechanism2"><h1>Click here for more info </h1></a></div>
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   <ul class="romanlist">
 
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    <li>Panozzo, C., Capuano, V., Fillinger, S. and Felenbok, B. (1997) ‘The zinc binuclear cluster Activator AlcR is able to bind to single sites but requires multiple repeated sites for synergistic activation of the alcA gene in Aspergillus nidulans’, <i>Journal of Biological Chemistry</i>, 272(36), pp. 22859–22865.  
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      <li style="text-align:left;"> Azevedo, A. M., Prazeres, D. M. F., Cabral, J. M., & Fonseca, L. P. (2005). Ethanol biosensors based on alcohol oxidase. <i>Biosensors and Bioelectronics</i>,21(2), 235-247.
 
     </li>
 
     </li>
 +
<li style="text-align: left;;"> Plants: Kinkema, M., Geijskes, R.J., Shand, K., Coleman, H.D., De Lucca, P.C., Palupe, A., Harrison, M.D., Jepson, I., Dale, J.L. and Sainz, M.B. (2013). An improved chemically inducible gene switch that functions in the monocotyledonous plant sugar cane. Plant Molecular Biology, 84(4-5), 443–454.
 +
</li>
 +
    <li style="text-align: left;;"> Panozzo, C., Capuano, V., Fillinger, S. and Felenbok, B. (1997). The zinc binuclear cluster Activator AlcR is able to bind to single sites but requires multiple repeated sites for synergistic activation of the alcA gene in Aspergillus nidulans. <i>Journal of Biological Chemistry</i>, 272(36), pp. 22859–22865.
 +
    </li>
 +
<li style="text-align: left;;"> Garoosi, A.G., Salter,M.G. , Caddick ,X.M and Tomsett, M.B. (2004). Characterization of the ethanol-inducible <i>alc</i> gene expression system in tomato. <i>Journal of experimental Botany</i>, 46 (416), pp. 1635-1642.
 +
</li>
 
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   </ul>
  
 
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<a class="projectlink" href="https://2016.igem.org/Team:Manchester"><< Main Page</a>
 
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{{Manchester/CSS/footer}}

Latest revision as of 14:39, 24 November 2016

Manchester iGEM 2016


Project Overview



Mechanism 1

Cell Free System
Mechanism 2 overview diagram

Enzymatic colourimetric assays are used to determine the concentration of a chemical in a solution by the conversion of a chromogen substrate into a coloured product. We have introduced a plasmid expressing recombinant Alcohol Oxidase 1 (AOx) from Pichia pastoris into Escherichia coli BL21 (DE3) strain that will then be used in the cell-free colorimetric system. This method involves the usage of AOx to oxidise ethanol, producing hydrogen peroxide (H2O2) as a by-product. H2O2 is used as an oxidising agent by horseradish peroxidase (HRP) to convert ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) to produce the colour change [1].



Click here for more info




Mechanism 2

Inducible Gene Switch



Mechanism 2 overview diagram

The alc gene expression system is one of the most reliable chemically inducible gene switches for use in plants [2] and fungus [3]. This system relies on the ability of AlcR, an alcohol-activated transcription factor, to bind to its target alcA promoter (PalcA). Based on this, we have engineered E. coli K-12 derivative DH5α and BL21 to induce expression of chromoproteins when AlcR binds to the native PalcA and variant of PalcA ( PalcA(var)) in the presence of ethanol [4].



Click here for more info

References

  • Azevedo, A. M., Prazeres, D. M. F., Cabral, J. M., & Fonseca, L. P. (2005). Ethanol biosensors based on alcohol oxidase. Biosensors and Bioelectronics,21(2), 235-247.
  • Plants: Kinkema, M., Geijskes, R.J., Shand, K., Coleman, H.D., De Lucca, P.C., Palupe, A., Harrison, M.D., Jepson, I., Dale, J.L. and Sainz, M.B. (2013). An improved chemically inducible gene switch that functions in the monocotyledonous plant sugar cane. Plant Molecular Biology, 84(4-5), 443–454.
  • Panozzo, C., Capuano, V., Fillinger, S. and Felenbok, B. (1997). The zinc binuclear cluster Activator AlcR is able to bind to single sites but requires multiple repeated sites for synergistic activation of the alcA gene in Aspergillus nidulans. Journal of Biological Chemistry, 272(36), pp. 22859–22865.
  • Garoosi, A.G., Salter,M.G. , Caddick ,X.M and Tomsett, M.B. (2004). Characterization of the ethanol-inducible alc gene expression system in tomato. Journal of experimental Botany, 46 (416), pp. 1635-1642.
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