Difference between revisions of "Team:Edinburgh/Basic Part"

 
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                       <li><a href="https://2015.igem.org/Team:Edinburgh/DNPBiosensor">DNP Biosensor</a></li>
 
                       <li><a href="https://2015.igem.org/Team:Edinburgh/DNPBiosensor">DNP Biosensor</a></li>
 
                       <li><a href="https://2015.igem.org/Team:Edinburgh/PMABiosensor">PMA Biosensor</a></li>
 
                       <li><a href="https://2015.igem.org/Team:Edinburgh/PMABiosensor">PMA Biosensor</a></li>
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                       <li><a href="https://2015.igem.org/Team:Edinburgh/CBD">Making it Stick</a></li>            
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                       <li><a href="https://2015.igem.org/Team:Edinburgh/CBD">Making it Stick</a></li>            
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                       <li><a href="https://2015.igem.org/Team:Edinburgh/Results">Results</a></li>
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                       <li><a href="https://2015.igem.org/Team:Edinburgh/Results">Limits of Detection</a></li>
 
                     </ul>
 
                     </ul>
 
                   </li>
 
                   </li>
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                     <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false">Parts<span class="caret"></span></a>
 
                     <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false">Parts<span class="caret"></span></a>
 
                     <ul class="dropdown-menu" role="menu">
 
                     <ul class="dropdown-menu" role="menu">
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                    <!-- <li><a href="https://2015.igem.org/Team:Edinburgh/Parts">Team Parts</a></li> -->
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                      <li><a href="https://2015.igem.org/Team:Edinburgh/Parts">Team Parts</a></li>  
 
                       <li><a href="https://2015.igem.org/Team:Edinburgh/Basic_Part">Basic Parts</a></li>
 
                       <li><a href="https://2015.igem.org/Team:Edinburgh/Basic_Part">Basic Parts</a></li>
 
                       <li><a href="https://2015.igem.org/Team:Edinburgh/Composite_Part">Composite Parts</a></li>
 
                       <li><a href="https://2015.igem.org/Team:Edinburgh/Composite_Part">Composite Parts</a></li>
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                    <!-- <li><a href="https://2015.igem.org/Team:Edinburgh/Part_Collection">Part Collection</a> </li> -->
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                      <li><a href="https://2015.igem.org/Team:Edinburgh/Part_Collection">Part Collection</a> </li>  
 
                       <li><a href="https://2015.igem.org/Team:Edinburgh/Improved_Part">Improved Parts</a></li>
 
                       <li><a href="https://2015.igem.org/Team:Edinburgh/Improved_Part">Improved Parts</a></li>
 
                       <li><a href="https://2015.igem.org/Team:Edinburgh/Characterisation_Part">Improved Characterisation</a></li>
 
                       <li><a href="https://2015.igem.org/Team:Edinburgh/Characterisation_Part">Improved Characterisation</a></li>
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                     </ul>
 
                     </ul>
 
                   </li>
 
                   </li>
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                   <li><a href="https://2015.igem.org/Team:Edinburgh/MedalCriteria">Medal Criteria</a></li>   
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                   <li><a href="https://2015.igem.org/Team:Edinburgh/MedalCriteria">Accomplishments</a></li>   
 
             </ul>
 
             </ul>
 
         </div>
 
         </div>
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                 <h1 class="brand-heading">Basic Parts</h1>
 
                 <h1 class="brand-heading">Basic Parts</h1>
 
                 <p class="intro-text">
 
                 <p class="intro-text">
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<br>
 
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<br>
 
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<br>
 
 
                 </p>
 
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                <div align="center">
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                    <a href="#accordion">
 +
                        <span class="arrowtext">Scroll down to read more</span>
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                        <img src="https://static.igem.org/mediawiki/2014/3/3e/Aalto_Helsinki_Nuoli.png" class="arrow">
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                    </a>
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                </div>
 
               </div>
 
               </div>
 
             </div>
 
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         <div class="panel-heading" role="tab" id="headingOne">
 
         <div class="panel-heading" role="tab" id="headingOne">
 
           <h4 class="panel-title">
 
           <h4 class="panel-title">
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             <a role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseOne" aria-expanded="false" aria-controls="collapseOne">
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             <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseOne" aria-expanded="false" aria-controls="collapseOne">
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               Heroin Esterase
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               Heroin Esterase BBa_K1615045
 
             </a>
 
             </a>
 
           </h4>
 
           </h4>
 
         </div>
 
         </div>
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         <div id="collapseOne" class="panel-collapse collapse in" role="tabpanel" aria-labelledby="headingOne">
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         <div id="collapseOne" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingOne">
 
           <div class="panel-body">
 
           <div class="panel-body">
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            <div class="col-md-6">
 
 
               <p style="color: black;">
 
               <p style="color: black;">
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              <h2>Materials</h2>
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            Heroin esterase, an acetylmorphine carboxylesterase,  was isolated from <i>Rhodococcus erythropolis</i> strain H1 in 1994 from the garden soil at Cambridge and is able to use heroin as its sole carbon and energy source by deacetylating the C-3 and C-6 groups to form morphine<sup>1</sup>. The gene <i>her</i> encodes this enzyme and can be expressed in the chassis <i>Escherichia coli</i><sup>2</sup>. The pH optimum for this enzyme is pH8.5 in bicine buffer<sup>1</sup>.
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              <ul>
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<br>
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                <li>1g Agarose
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<br>  
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                <li>100ml 1X TAE buffer
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<img src="https://static.igem.org/mediawiki/2015/b/b1/Edigem15_bparts_her1.jpg" class="img-responsive">
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                <li>5µl GelRed stain
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<br>
 +
<br>
 +
The activity of heroin esterase can be tested using 4-nitrophenyl acetate which is hydrolysed by heroin esterase to form 4-nitrophenol and acetate<sup>3</sup>. This produces a yellow colour which can be read at 410 nm.
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<br>
 +
<img src="https://static.igem.org/mediawiki/2015/c/cc/Edigem15_bparts_her2.jpg" class="img-responsive">
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<br>
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 +
<img src="https://static.igem.org/mediawiki/2015/c/c1/Hertab1.png" class="img-responsive">
 +
<br>
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<img src="https://static.igem.org/mediawiki/2015/0/07/Hertab2.png" class="img-responsive">
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<br>
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<img src="https://static.igem.org/mediawiki/2015/9/96/Herest1.jpg" class="img-responsive">
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<br>
 +
<b>Design:</b> The sequence for our enzyme used the original sequence from Rathbone, et al.<sup>2</sup>, which was then codon optimised for <i>E. coli</i>. The RFC25 prefix and suffix were added which required all illegal sites (EcoRI, SpeI, AgeI, NotI, NgoMIV and XbaI) to be removed. As this was a difficult sequence to make as a gBlock, it was ordered as a gene in an ampicillin backbone where it was then digested and ligated into the pSB1C3 backbone.
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<br>
 +
<br>
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<br>
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<br>
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<sup>1</sup>Cameron, G. W., Jordan, K. N., Holt, P. J., Baker, P. B., Lowe, C. R., & Bruce, N. C. (1994). Identification of a heroin esterase in Rhodococcus sp. strain H1. Applied and environmental microbiology, 60(10), 3881-3883.
 +
<br>
 +
<br><sup>2</sup>Rathbone, D. A., Holt, P. J., Lowe, C. R., & Bruce, N. C. (1997). Molecular analysis of the Rhodococcus sp. strain H1 her gene and characterization of its product, a heroin esterase, expressed in Escherichia coli. <i>Applied and environmental microbiology</i>, 63(5), 2062-2066.
 +
<br>
 +
<br>
 +
<sup>3</sup>Sigma-aldrich. 4-nitrophenyl acetate product information.
 +
 
 +
 
 
               </ul>
 
               </ul>
 
             </p>
 
             </p>
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            </div>
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            <div class="col-md-6">
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            <div align="center">
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              <p class="text-muted">
+
                <a href="#" class="btn btn-primary btn-lg outline" role="button">Check it out in the registry</a>
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              <h2>Procedure</h2>
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            </div>
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              <ul>
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−
                <li>1. Mix the agarose with the 1X TAE buffer in a flask.
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                <li>2. Heat the mixture until all the agarose is dissolved.
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                <li>3. Swirl the flask under cold running water to cool the mixture.
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                <li> 4. Add the gel stain.
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                <li>5. Pour into an assembled gel tray and let it cool.
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              </uL>
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            </p>
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             </div>
 
             </div>
 
           </div>
 
           </div>
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           <h4 class="panel-title">
 
           <h4 class="panel-title">
 
             <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseTwo" aria-expanded="false" aria-controls="collapseTwo">
 
             <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseTwo" aria-expanded="false" aria-controls="collapseTwo">
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             Morphine Dehydrogenase
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             Morphine-6-Dehydrogenase BBa_K1615000
 
             </a>
 
             </a>
 
           </h4>
 
           </h4>
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         <div id="collapseTwo" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingTwo">
 
         <div id="collapseTwo" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingTwo">
 
           <div class="panel-body">
 
           <div class="panel-body">
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            <div class="col-md-6">
 
 
               <p>
 
               <p>
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              <h2>Materials</h2>
+
              The structural gene morphine-6-dehydrogenase (<i>morA</i>) was first isolated from <i>Pseudomonas putida</i> M10 as it is capable of growth with morphine as its sole carbon source<sup>1</sup>. Morphine dehydrogenase (MDH) catalyses the oxidation of both morphine and codeine to produce morphinone and codeinone respectively. During this process NADP<sup>+</sup> is reduced to NADPH which means that this enzyme is frequently used to detect morphine and codeine<sup>2</sup>.
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              <ul>
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<br>
−
                <li>1% Agarose
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<br>
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                <li>1X TAE buffer
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<img src="https://static.igem.org/mediawiki/2015/4/4f/Morphine_dehydrogenase_activity.jpeg" class="img-responsive">
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                <li>5X loading dye
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<br>
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                <li>DNA ladder
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<br>
−
                <li>DNA samples
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<br>
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              </ul>
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<br>To test the morphine dehydrogenase activity it can be coupled with morphine and NADP<sup>+</sup> to produce morphinone and NADPH. The amount of NADPH produced can be measured at 340nm. Morphine dehydrogenase with t7 promoter characterised by Edinburgh iGEM team was provided by Prof Chris French. Michealis Menten kinetic analysis was performed giving values of Vmax and Km, 61.22 and 140.5 uM respectively.
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            </p>
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<br>
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            </div>
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<img src="https://static.igem.org/mediawiki/2015/2/2f/Morpg.jpg" class="img-responsive">
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            <div class="col-md-6">
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<br>
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              <p>
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<br>Following table summarises the kinetic analysis and statistics of the measurment.
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              <h2>Procedure</h2>
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<img src="https://static.igem.org/mediawiki/2015/e/e1/Morphgraph.jpg" class="img-responsive">
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              <ul>
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<br>
−
                <li>1. Place gel tray into the electrophoresis apparatus.
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<br>
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                <li>2. Pour 1X TAE so that the gel is covered by buffer.
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<b>Design:</b> To make this gene standardised it was codon optimised for the chassis <i>Escherichia coli</i> as well as making it RFC25 compatible which required removing all illegal restriction sites in the gene sequence.
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                <li>3. Prepare the samples by adding the appropriate amount of loading dye.
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<br>
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                <li>4. Load samples and DNA ladder into wells on the gel.
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<br>
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                <li>5. Run the gel at roughly 100V for around an hour
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<br>
 +
<br>
 +
<sup>1</sup>Bruce, N. C., Wilmot, C. J., Jordan, K. N., Trebilcock, A. E., Stephens, L. D. G., & Lowe, C. R. (1990). Microbial degradation of the morphine alkaloids: identification of morphinone as an intermediate in the metabolism of morphine by Pseudomonas putida M10. <i>Archives of microbiology</i>, 154(5), 465-470.
 +
<br><sup>2</sup>Rathbone, D. A., Holt, P. J., Lowe, C. R., & Bruce, N. C. (1997). Molecular analysis of the Rhodococcus sp. strain H1 her gene and characterization of its product, a heroin esterase, expressed in Escherichia coli. <i>Applied and environmental microbiology</i>, 63(5), 2062-2066.
 +
<br><sup>2</sup>WALKER, E., et al. "Mechanistic studies of morphine dehydrogenase and stabilization against covalent inactivation." Biochem. J 345 (2000): 687-692.
  
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              </uL>
 
 
             </p>
 
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            </div>
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            <div align="center">
 +
                <a href="#" class="btn btn-primary btn-lg outline" role="button">Check it out in the registry</a>
 +
            </div>
 +
           
 
           </div>
 
           </div>
 
         </div>
 
         </div>
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           <h4 class="panel-title">
 
           <h4 class="panel-title">
 
             <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseThree" aria-expanded="false" aria-controls="collapseThree">
 
             <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseThree" aria-expanded="false" aria-controls="collapseThree">
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               TVEL5 Laccase
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               Monoamine oxidase A BBa_K1615022
 
             </a>
 
             </a>
 
           </h4>
 
           </h4>
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         <div id="collapseThree" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingThree">
 
         <div id="collapseThree" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingThree">
 
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               <p>
 
               <p>
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              <h2>Materials</h2>
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                Monoamine oxidase A is coded by the gene <i>maoA</i> and is subject to catabolite and ammonium ion repression<sup>1</sup>. Amine oxidases that contain copper/topaquinone (TPQ), like monoamine oxidase A, convert primary amines into their corresponding aldehydes, hydrogen peroxide and ammonia<sup>2</sup>.
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              <ul>
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<br>
−
                <li>10ml Luria Broth (LB)
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<br>
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                <li>10µl Specific Antibiotic at 1000x (Chloramphenicol, Ampicillin or Kanamycin)
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To test the activity of monoamine oxidase A, tyramine can be used as a substrate and its corresponding aldehyde as well as ammonia and hydrogen peroxide will be produced. When the hydrogen peroxide is coupled with horseradish peroxidase and Amplex red, resorufin, a red colour, will be produced.
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                <li>Loop (for picking colony)
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<br>
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                <li>Ethanol
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<br>
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              </ul>
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Design: This monoamine oxidase A sequence was found in <i>Klebsiella pneumoniae</i><sup>3</sup> and was codon optimised for the chassis Escherichia coli as well as made RFC25 compatible with the corresponding prefix and suffix and illegal restriction sites were removed.
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            </p>
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<br>
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            </div>
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<br>
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            <div class="col-md-6">
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<sup>1</sup>Oka, M., Murooka, Y., & Harada, T. (1980). Genetic control of tyramine oxidase, which is involved in derepressed synthesis of arylsulfatase in Klebsiella aerogenes. <i>Journal of bacteriology</i>, 143(1), 321-327.
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              <p>
+
<br><sup>2</sup>McIntire, W. S., & Hartmann, C. (1993). Copper-containing amine oxidases. <i>Principles and applications of quinoproteins</i>, 97-171.
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              <h2>Procedure</h2>
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<br><sup>3</sup>Sugino, H., Sasaki, M., Azakami, H., Yamashita, M., & Murooka, Y. (1992). A monoamine-regulated Klebsiella aerogenes operon containing the monoamine oxidase structural gene (maoA) and the maoC gene. <i>Journal of bacteriology</i>, 174(8), 2485-2492.
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              <ul>
+
 
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                <li>1. Pour 10ml of LB into a 50ml Falcon tube.  
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                <li>2. Pipette 10µl of antibiotic into the broth.
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                <li>3. Dip loop in ethanol and flame to sterilise. Once it is cool, pick colony and transfer to a 50ml Falcon tube.
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                <li>4. Incubate at 37°C overnight in a shaking incubator.
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−
              </uL>
+
 
             </p>
 
             </p>
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            </div>
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            <div align="center">
 +
                <a href="#" class="btn btn-primary btn-lg outline" role="button">Check it out in the registry</a>
 +
            </div>
 +
         
 
           </div>
 
           </div>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
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      <div class="panel panel-default">
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        <div class="panel-heading" role="tab" id="headingFour">
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          <h4 class="panel-title">
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            <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseFour" aria-expanded="false" aria-controls="collapseFour">
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            Monoamine Oxidase A
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            </a>
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          </h4>
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        </div>
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        <div id="collapseFour" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingFour">
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          <div class="panel-body">
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            <div class="col-md-6">
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              <p>
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              <h2>Materials</h2>
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              <ul>
+
−
                <li>Buffer QG
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−
                <li>10µl 3M sodium acetate
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−
                <li>Isopropanol
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−
                <li>750µl Buffer PE
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                <li>25µl Buffer EB
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              </ul>
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            </p>
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            </div>
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            <div class="col-md-6">
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              <p>
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−
              <h2>Procedure</h2>
+
−
              All centrifuge steps are carried out at 13,000 rpm.
+
−
              <ul>
+
−
                <li>1. Excise the region of gel containing the DNA fragment using a scalpel.  Cut close to the DNA to minimise the gel volume.
+
−
                <li>2. Place the gel slice in a 1.5ml tube and weigh it. Record the volume of the gel.
+
−
                <li>3.  Add 300µl of Buffer QG for each 100mg of gel.
+
−
                <li>4. Incubate at 50°C for 10 minutes or until the gel has completely dissolved. Mix by vortexing the tube every 2 minutes during the incubation.
+
−
                <li>5. Once the gel is completely dissolved, the mixture should be yellow. If the mixture is orange or violet add 10 µl of 3M sodium acetate and mix until it turns yellow. Yellow colour indicates the solution is the optimum pH for DNA binding to the QIAquick membrane.
+
−
                <li>6. Add 1 gel volume of isopropanol to the solution and mix (1:1 volumes of isopropanol to gel slice).
+
−
                <li>7. Place a QIAquick spin column in a 2ml collection tube.
+
−
                <li>8. Pipette the sample onto the QIAquick column and centrifuge. Discard flow-through.
+
−
                <li>9. Place column back in same collection tube. Add 500µl of Buffer QG to the column and centrifuge for 1 minute to remove all traces of agarose.
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                <li>10. Wash column by adding 750µl buffer PE. Let it stand for 2-5 min and then centrifuge for 1 minute.
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                <li>11. Discard the flow-through. Centrifuge for 1 minute to remove the residual buffer PE.
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                <li>12. Then place the column in a clean, labelled 1.5ml Eppendorf tube.
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                <li>13. To elute the DNA, add 25µl of Buffer EB to the centre of the column membrane, let it stand for 1 minute and then centrifuge for 1 minute.
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                <li>14. Using a pipette, transfer the flow-through back into the centre of the column. Let it stand for 1 minute and then centrifuge for 1 minute. The DNA will now be in the flow-through.
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Latest revision as of 18:54, 20 November 2015

Basic Parts

Heroin esterase, an acetylmorphine carboxylesterase, was isolated from Rhodococcus erythropolis strain H1 in 1994 from the garden soil at Cambridge and is able to use heroin as its sole carbon and energy source by deacetylating the C-3 and C-6 groups to form morphine1. The gene her encodes this enzyme and can be expressed in the chassis Escherichia coli2. The pH optimum for this enzyme is pH8.5 in bicine buffer1.



The activity of heroin esterase can be tested using 4-nitrophenyl acetate which is hydrolysed by heroin esterase to form 4-nitrophenol and acetate3. This produces a yellow colour which can be read at 410 nm.




Design: The sequence for our enzyme used the original sequence from Rathbone, et al.2, which was then codon optimised for E. coli. The RFC25 prefix and suffix were added which required all illegal sites (EcoRI, SpeI, AgeI, NotI, NgoMIV and XbaI) to be removed. As this was a difficult sequence to make as a gBlock, it was ordered as a gene in an ampicillin backbone where it was then digested and ligated into the pSB1C3 backbone.



1Cameron, G. W., Jordan, K. N., Holt, P. J., Baker, P. B., Lowe, C. R., & Bruce, N. C. (1994). Identification of a heroin esterase in Rhodococcus sp. strain H1. Applied and environmental microbiology, 60(10), 3881-3883.

2Rathbone, D. A., Holt, P. J., Lowe, C. R., & Bruce, N. C. (1997). Molecular analysis of the Rhodococcus sp. strain H1 her gene and characterization of its product, a heroin esterase, expressed in Escherichia coli. Applied and environmental microbiology, 63(5), 2062-2066.

3Sigma-aldrich. 4-nitrophenyl acetate product information.

The structural gene morphine-6-dehydrogenase (morA) was first isolated from Pseudomonas putida M10 as it is capable of growth with morphine as its sole carbon source1. Morphine dehydrogenase (MDH) catalyses the oxidation of both morphine and codeine to produce morphinone and codeinone respectively. During this process NADP+ is reduced to NADPH which means that this enzyme is frequently used to detect morphine and codeine2.





To test the morphine dehydrogenase activity it can be coupled with morphine and NADP+ to produce morphinone and NADPH. The amount of NADPH produced can be measured at 340nm. Morphine dehydrogenase with t7 promoter characterised by Edinburgh iGEM team was provided by Prof Chris French. Michealis Menten kinetic analysis was performed giving values of Vmax and Km, 61.22 and 140.5 uM respectively.


Following table summarises the kinetic analysis and statistics of the measurment.

Design: To make this gene standardised it was codon optimised for the chassis Escherichia coli as well as making it RFC25 compatible which required removing all illegal restriction sites in the gene sequence.



1Bruce, N. C., Wilmot, C. J., Jordan, K. N., Trebilcock, A. E., Stephens, L. D. G., & Lowe, C. R. (1990). Microbial degradation of the morphine alkaloids: identification of morphinone as an intermediate in the metabolism of morphine by Pseudomonas putida M10. Archives of microbiology, 154(5), 465-470.
2Rathbone, D. A., Holt, P. J., Lowe, C. R., & Bruce, N. C. (1997). Molecular analysis of the Rhodococcus sp. strain H1 her gene and characterization of its product, a heroin esterase, expressed in Escherichia coli. Applied and environmental microbiology, 63(5), 2062-2066.
2WALKER, E., et al. "Mechanistic studies of morphine dehydrogenase and stabilization against covalent inactivation." Biochem. J 345 (2000): 687-692.

Monoamine oxidase A is coded by the gene maoA and is subject to catabolite and ammonium ion repression1. Amine oxidases that contain copper/topaquinone (TPQ), like monoamine oxidase A, convert primary amines into their corresponding aldehydes, hydrogen peroxide and ammonia2.

To test the activity of monoamine oxidase A, tyramine can be used as a substrate and its corresponding aldehyde as well as ammonia and hydrogen peroxide will be produced. When the hydrogen peroxide is coupled with horseradish peroxidase and Amplex red, resorufin, a red colour, will be produced.

Design: This monoamine oxidase A sequence was found in Klebsiella pneumoniae3 and was codon optimised for the chassis Escherichia coli as well as made RFC25 compatible with the corresponding prefix and suffix and illegal restriction sites were removed.

1Oka, M., Murooka, Y., & Harada, T. (1980). Genetic control of tyramine oxidase, which is involved in derepressed synthesis of arylsulfatase in Klebsiella aerogenes. Journal of bacteriology, 143(1), 321-327.
2McIntire, W. S., & Hartmann, C. (1993). Copper-containing amine oxidases. Principles and applications of quinoproteins, 97-171.
3Sugino, H., Sasaki, M., Azakami, H., Yamashita, M., & Murooka, Y. (1992). A monoamine-regulated Klebsiella aerogenes operon containing the monoamine oxidase structural gene (maoA) and the maoC gene. Journal of bacteriology, 174(8), 2485-2492.