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

 
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                   <a href="https://2015.igem.org/Team:Edinburgh">Home</a></li>
 
                   <a href="https://2015.igem.org/Team:Edinburgh">Home</a></li>
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                    <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false">About<span class="caret"></span></a>
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                      <li><a href="https://2015.igem.org/Team:Edinburgh/Team">Team</a></li>
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                       <li><a href="https://2015.igem.org/Team:Edinburgh/Description">Description</a></li>
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                       <li><a href="https://2015.igem.org/Team:Edinburgh/Description">Overview</a></li>
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                      <li><a href="https://2015.igem.org/Team:Edinburgh/Experiments">Experiments</a> </li>
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                    <!-- <li><a href="https://2015.igem.org/Team:Edinburgh/Experiments">Experiments</a> </li> -->
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                       <li><a href="https://2015.igem.org/Team:Edinburgh/Project/Protocols">Protocols</a> </li>
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                       <li><a href="https://2015.igem.org/Team:Edinburgh/HeroinBiosensor">Heroin Biosensor</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/DNPBiosensor">DNP Biosensor</a></li>
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                       <li><a href="https://2015.igem.org/Team:Edinburgh/Design">Design</a></li>
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                       <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/Results">Limits of Detection</a></li>
 
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                     <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false">Parts<span class="caret"></span></a>
 
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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>
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                      <li><a href="https://2015.igem.org/Team:Edinburgh/Characterisation_Part">Improved Characterisation</a></li>
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                    <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false">Dry Lab<span class="caret"></span></a>
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                      <li><a href="https://2015.igem.org/Team:Edinburgh/Modeling">Modeling</a></li>
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                      <li><a href="https://2015.igem.org/Team:Edinburgh/Design">Design</a></li>
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                      <li><a href="https://2015.igem.org/Team:Edinburgh/Software">Software</a></li>
 
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                  <li><a href="https://2015.igem.org/Team:Edinburgh/Attributions">Attributions</a></li>
 
 
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                     <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false">Policy and Practices<span class="caret"></span></a>
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                       <li><a href="https://2015.igem.org/Team:Edinburgh/Practices">Our Story</a></li>
 
                       <li><a href="https://2015.igem.org/Team:Edinburgh/Practices">Our Story</a></li>
 
                       <li><a href="https://2015.igem.org/Team:Edinburgh/Safety">Safety</a> </li>
 
                       <li><a href="https://2015.igem.org/Team:Edinburgh/Safety">Safety</a> </li>
 
                       <li><a href="https://2015.igem.org/Team:Edinburgh/Legality">Legality</a> </li>
 
                       <li><a href="https://2015.igem.org/Team:Edinburgh/Legality">Legality</a> </li>
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                      <li><a href="https://2015.igem.org/Team:Edinburgh/Outreach">Outreach</a> </li>
 
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                  <li><a href="https://2015.igem.org/Team:Edinburgh/Modeling">Modeling</a></li>
 
 
                   <li class="dropdown">
 
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                     <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false">InterLab<span class="caret"></span></a>
 
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                  <li><a href="software.html">Software</a></li>
 
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                  <li><a href="entrepreneurship.html">Entrepreneurship</a></li>
 
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                  <li><a href="collaborations.html">Collaborations</a></li>
 
 
                   <li class="dropdown">
 
                   <li class="dropdown">
 
                     <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false">Notebook<span class="caret"></span></a>
 
                     <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false">Notebook<span class="caret"></span></a>
 
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                       <li><a href="https://2015.igem.org/Team:Edinburgh/InterLab">Heroin Purity</a></li>
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                       <li><a href="https://2015.igem.org/Team:Edinburgh/Project/Protocols">Protocols</a></li>
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                       <li><a href="https://2015.igem.org/Team:Edinburgh/InterLabBook">PMA Detection</a> </li>
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                      <li><a href="https://2015.igem.org/Team:Edinburgh/Notebook/HeroinPurity">Heroin Purity</a></li>
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                       <li><a href="https://2015.igem.org/Team:Edinburgh/InterLabBook">DNP Detection</a> </li>
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                       <li><a href="https://2015.igem.org/Team:Edinburgh/Notebook/PMADetection">PMA Detection</a> </li>
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                       <li><a href="https://2015.igem.org/Team:Edinburgh/InterLabBook">Fluid Dynamics</a> </li>
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                       <li><a href="https://2015.igem.org/Team:Edinburgh/Notebook/DNPDetection">DNP Detection</a> </li>
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                       <li><a href="https://2015.igem.org/Team:Edinburgh/Notebook/FluidDynamics">Fluid Dynamics</a> </li>
 
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                </ul>
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                  <li><a href="https://2015.igem.org/Team:Edinburgh/MedalCriteria">Accomplishments</a></li>  
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         <header class="intro">
 
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                 <h1 class="brand-heading">Basic Parts</h1>
 
                 <h1 class="brand-heading">Basic Parts</h1>
 
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                    <a href="#accordion">
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                        <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>
 
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               1% Agarose Gel
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               Heroin Esterase BBa_K1615045
 
             </a>
 
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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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                <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>
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<br>
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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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<img src="https://static.igem.org/mediawiki/2015/c/cc/Edigem15_bparts_her2.jpg" class="img-responsive">
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<img src="https://static.igem.org/mediawiki/2015/c/c1/Hertab1.png" class="img-responsive">
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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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<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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<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.
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<br>
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<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.
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<sup>3</sup>Sigma-aldrich. 4-nitrophenyl acetate product information.
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                <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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                <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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             Agarose Gel Electrophoresis
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             Morphine-6-Dehydrogenase BBa_K1615000
 
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              <h2>Materials</h2>
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              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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                <li>DNA samples
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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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<br>
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            </div>
+
<img src="https://static.igem.org/mediawiki/2015/2/2f/Morpg.jpg" class="img-responsive">
−
            <div class="col-md-6">
+
<br>
−
              <p>
+
<br>Following table summarises the kinetic analysis and statistics of the measurment.
−
              <h2>Procedure</h2>
+
<img src="https://static.igem.org/mediawiki/2015/e/e1/Morphgraph.jpg" class="img-responsive">
−
              <ul>
+
<br>
−
                <li>1. Place gel tray into the electrophoresis apparatus.
+
<br>
−
                <li>2. Pour 1X TAE so that the gel is covered by buffer.
+
<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.
−
                <li>3. Prepare the samples by adding the appropriate amount of loading dye.
+
<br>
−
                <li>4. Load samples and DNA ladder into wells on the gel.
+
<br>
−
                <li>5. Run the gel at roughly 100V for around an hour
+
<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.
  
−
              </uL>
 
 
             </p>
 
             </p>
−
            </div>
+
            <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>
 
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             <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseThree" aria-expanded="false" aria-controls="collapseThree">
−
               Culture
+
               Monoamine oxidase A BBa_K1615022
 
             </a>
 
             </a>
 
           </h4>
 
           </h4>
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          <div class="col-md-6">
 
 
               <p>
 
               <p>
−
              <h2>Materials</h2>
+
                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>.
−
              <ul>
+
<br>
−
                <li>10ml Luria Broth (LB)
+
<br>
−
                <li>10µl Specific Antibiotic at 1000x (Chloramphenicol, Ampicillin or Kanamycin)
+
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.
−
                <li>Loop (for picking colony)
+
<br>
−
                <li>Ethanol
+
<br>
−
              </ul>
+
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.
 +
<br>
 +
<br>
 +
<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.
 +
<br><sup>2</sup>McIntire, W. S., & Hartmann, C. (1993). Copper-containing amine oxidases. <i>Principles and applications of quinoproteins</i>, 97-171.
 +
<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.
 +
 
 
             </p>
 
             </p>
−
            </div>
+
            <div align="center">
−
            <div class="col-md-6">
+
                <a href="#" class="btn btn-primary btn-lg outline" role="button">Check it out in the registry</a>
−
              <p>
+
            </div>
−
              <h2>Procedure</h2>
+
         
−
              <ul>
+
−
                <li>1. Pour 10ml of LB into a 50ml Falcon tube.
+
−
                <li>2. Pipette 10µl of antibiotic into the broth.
+
−
                <li>3. Dip loop in ethanol and flame to sterilise. Once it is cool, pick colony and transfer to a 50ml Falcon tube.
+
−
                <li>4. Incubate at 37°C overnight in a shaking incubator.
+
−
              </uL>
+
−
            </p>
+
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            </div>
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           </div>
 
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         </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">
+
−
            Gel Purification using QIAquick Gel Extraction Kit
+
−
            </a>
+
−
          </h4>
+
−
        </div>
+
−
        <div id="collapseFour" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingFour">
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          <div class="panel-body">
+
−
            <div class="col-md-6">
+
−
              <p>
+
−
              <h2>Materials</h2>
+
−
              <ul>
+
−
                <li>Buffer QG
+
−
                <li>10µl 3M sodium acetate
+
−
                <li>Isopropanol
+
−
                <li>750µl Buffer PE
+
−
                <li>25µl Buffer EB
+
−
              </ul>
+
−
            </p>
+
−
            </div>
+
−
            <div class="col-md-6">
+
−
              <p>
+
−
              <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.
+
−
                <li>10. Wash column by adding 750µl buffer PE. Let it stand for 2-5 min and then centrifuge for 1 minute.
+
−
                <li>11. Discard the flow-through. Centrifuge for 1 minute to remove the residual buffer PE.
+
−
                <li>12. Then place the column in a clean, labelled 1.5ml Eppendorf tube.
+
−
                <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.
+
−
                <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.
+
−
              </uL>
+
−
            </p>
+
−
            </div>
+
−
          </div>
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        </div>
+
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      </div>
+
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      <div class="panel panel-default">
+
−
        <div class="panel-heading" role="tab" id="headingFive">
+
−
          <h4 class="panel-title">
+
−
            <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseFive" aria-expanded="false" aria-controls="collapseFive">
+
−
            Glycerol Stock
+
−
            </a>
+
−
          </h4>
+
−
        </div>
+
−
        <div id="collapseFive" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingFive">
+
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          <div class="panel-body">
+
−
            <div class="col-md-6">
+
−
              <p>
+
−
              <h2>Materials</h2>
+
−
              <ul>
+
−
                <li>500µl 50% glycerol solution
+
−
                <li>500µl cultured cells
+
−
              </ul>
+
−
            </p>
+
−
            </div>
+
−
            <div class="col-md-6">
+
−
              <p>
+
−
              <h2>Procedure</h2>
+
−
              <ul>
+
−
                <li>1. Add the 50% glycerol to a sterile Eppendorf tube.
+
−
                <li>2. Add 500µl of cells to the tube and vortex to mix.
+
−
                <li>3. Freeze at -80°C.
+
−
              </uL>
+
−
            </p>
+
−
            </div>
+
−
          </div>
+
−
        </div>
+
−
      </div>
+
−
      <div class="panel panel-default">
+
−
        <div class="panel-heading" role="tab" id="headingSix">
+
−
          <h4 class="panel-title">
+
−
            <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseSix" aria-expanded="false" aria-controls="collapseSix">
+
−
            Heat Shock Transformation
+
−
            </a>
+
−
          </h4>
+
−
        </div>
+
−
        <div id="collapseSix" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingSix">
+
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          <div class="panel-body">
+
−
            <div class="col-md-6">
+
−
              <p>
+
−
              <h2>Materials</h2>
+
−
              <ul>
+
−
                <li>2.5µl ligated DNA or 0.5µl purified plasmid
+
−
                <li>50µl chemically competent DH5α E.coli cells
+
−
                <li>250µl Luria Broth
+
−
                <li>Petri dish with LB agar and specific antibiotic (chloramphenicol or ampicillin)
+
−
              </ul>
+
−
            </p>
+
−
            </div>
+
−
            <div class="col-md-6">
+
−
              <p>
+
−
              <h2>Procedure</h2>
+
−
              <ul>
+
−
                <li>1. Thaw the competent cells on ice.
+
−
                <li>2. Add 50µl of cells to a pre-chilled, labelled microcentrifuge tube.
+
−
                <li>3. Add 2.5µl of DNA suspension to the tube. Pipette up and down to mix.
+
−
                <li>4. Incubate tube on ice for 30 min.
+
−
                <li>5. Heat shock in a water bath at 42°C for 60s.
+
−
                <li>6. Place tube back on ice for 3 min.
+
−
                <li>7. Add 250µl of LB to the tube.
+
−
                <li>8. Incubate the tube at 37°C for 1 hour.
+
−
                <li>9. Prepare two plates for each transformation, one plated with 200µl of cells and another plated with 100µl of cells.
+
−
                <li>10. Incubate at 37°C overnight (12-14 hours).
+
−
              </uL>
+
−
            </p>
+
−
            </div>
+
−
          </div>
+
−
        </div>
+
−
      </div>
+
−
      <div class="panel panel-default">
+
−
        <div class="panel-heading" role="tab" id="headingSeven">
+
−
          <h4 class="panel-title">
+
−
            <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseSeven" aria-expanded="false" aria-controls="collapseSeven">
+
−
            Miniprep using QIAprep Spin Miniprep Kit
+
−
            </a>
+
−
          </h4>
+
−
        </div>
+
−
        <div id="collapseSeven" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingSeven">
+
−
          <div class="panel-body">
+
−
            <div class="col-md-6">
+
−
              <p>
+
−
              <h2>Materials</h2>
+
−
              <ul>
+
−
                <li>10ml cell culture
+
−
                <li>250µl Buffer P1
+
−
                <li>250µl Buffer P2
+
−
                <li>350µl Buffer N3
+
−
                <li>750µl Buffer PE
+
−
                <li>50µl Buffer EB
+
−
              </ul>
+
−
            </p>
+
−
            </div>
+
−
            <div class="col-md-6">
+
−
              <p>
+
−
              <h2>Procedure</h2>
+
−
              <ul>
+
−
                <li>1. Centrifuge 10ml cell culture at 4,500 x g for 5 minutes and pour off the supernatant. Centrifuge at 4,500 x g for 2 minutes and remove the supernatant by pipetting.
+
−
                <li>2. Resuspend pelleted cells in 250 µl Buffer P1 and transfer the solution to a labelled Eppendorf tube.
+
−
                <li>3. Add 250 µl Buffer P2 and gently invert the tube 4–6 times to mix. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 minutes.
+
−
                <li>4. Add 350 µl Buffer N3 and invert the tube immediately but gently 4–6 times. The solution should become cloudy.
+
−
                <li>5. Centrifuge for 10 min at 13,000 rpm in an Eppendorf tube. A compact white pellet will form.
+
−
                <li>6. Apply the supernatant to the QIAprep Spin Column by pipetting.
+
−
                <li>7. Centrifuge for 60s at 13,000 rpm. Discard the flow-through.
+
−
                <li>8. Wash QIAprep Spin Column by adding 750µl Buffer PE and centrifuging for 60s at 13,000 rpm.
+
−
                <li>9. Discard the flow-through, and centrifuge for an additional 60s at 13,000 rpm to remove residual wash buffer.
+
−
                <li>10. Place the QIAprep column in a clean, labelled Eppendorf tube. To double elute DNA, add 50 µl Buffer EB to the centre of each QIAprep Spin Column, let stand for 1 min, and centrifuge for 1 min at 13,000 rpm. Re-apply flow-through to the centre of the QIAprep Spin Column, let stand for 1 min and centrifuge for 1 min at 13,000 rpm.
+
−
                <li>11. Discard column. Flow-through contains the DNA.
+
−
              </uL>
+
−
            </p>
+
−
            </div>
+
−
          </div>
+
−
        </div>
+
−
      </div>
+
−
      <div class="panel panel-default">
+
−
        <div class="panel-heading" role="tab" id="headingEight">
+
−
          <h4 class="panel-title">
+
−
            <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseEight" aria-expanded="false" aria-controls="collapseEight">
+
−
            Nanodrop
+
−
            </a>
+
−
          </h4>
+
−
        </div>
+
−
        <div id="collapseEight" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingEight">
+
−
          <div class="panel-body">
+
−
            <div class="col-md-6">
+
−
              <p>
+
−
              <h2>Materials</h2>
+
−
              <ul>
+
−
                <li>Buffer EB to blank
+
−
                <li>DNA sample
+
−
              </ul>
+
−
            </p>
+
−
            </div>
+
−
            <div class="col-md-6">
+
−
              <p>
+
−
              <h2>Procedure</h2>
+
−
              <ul>
+
−
                <li>1. Wipe the NanoDrop 2000 clean before pipetting 1µl of buffer EB and lowering the arm. Blank the NanoDrop spectrophotometer.
+
−
                <li>2. Clean the buffer EB from the pedestal and apply 1µl of sample. Measure and record the absorbance, and repeat for remainder of samples.
+
−
                <li>3. Clean the NanoDrop pedestal after use.
+
−
              </uL>
+
−
            </p>
+
−
            </div>
+
−
          </div>
+
−
        </div>
+
−
      </div>
+
−
            <div class="panel panel-default">
+
−
        <div class="panel-heading" role="tab" id="headingNine">
+
−
          <h4 class="panel-title">
+
−
            <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseNine" aria-expanded="false" aria-controls="collapseNine">
+
−
            PCR Purification using QIAquick PCR Purification Kit
+
−
            </a>
+
−
          </h4>
+
−
        </div>
+
−
        <div id="collapseNine" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingNine">
+
−
          <div class="panel-body">
+
−
            <div class="col-md-6">
+
−
              <p>
+
−
              <h2>Materials</h2>
+
−
              <ul>
+
−
                <li>Buffer PB
+
−
                <li>10µl of 3M sodium acetate
+
−
                <li>750µl buffer PE
+
−
                <li>25µl buffer EB
+
−
              </ul>
+
−
            </p>
+
−
            </div>
+
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            <div class="col-md-6">
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              All centrifugation steps are carried out at 17,900 x g (13,000 rpm)
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                <li>1. Add 5 volumes of buffer PB to 1 volume of PCR sample (e.g. 500µl PB to 100µl PCR sample) and mix.
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                <li>3. Place a QIAquick spin column into a 2ml collection tube.
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                <li>5. Discard the flow-through and replace the column in the same collection tube.
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                <li>6. Add 750µl of buffer PE to the column and centrifuge for 60s.
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                <li>7. Discard the flow-through, replace the column in the collection tube and centrifuge for a further 60s to remove residual buffer.
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                <li>8. Place the column in a clean 1.5ml Eppendorf tube.
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                <li>9. Elute the DNA by adding 25µl buffer EB to the centre of the membrane in the column. Let it stand for 1 minute then centrifuge for 1 minute.
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                <li>10. 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.
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                <li>11. The purified DNA is now present in the flow-through.
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            Resuspending gBlocks
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              <h2>Materials</h2>
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                <li>gBlocks dry gene fragment
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                <li>1. Centrifuge the tube containing the gBlocks gene fragment pellet at 3000 x g for 5s.
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                <li>3. Vortex to mix.
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                <li>5. Incubate at 50°C for 20 mins.
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            Sequencing
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              <h2>Materials</h2>
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                <li>200-500ng DNA sample
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                <li>0.32µl 10µM Vf2 and Vr primers
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                <li>1. Prepare the DNA for sequencing by adding DNA, primers and water to a final volume of 6µl.
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                <li>3. Send the samples to be Sanger sequenced by Edinburgh Genomics using an ABI 3730 DNA analyser.
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                <li>4. Edinburgh Genomics will send an email with the sequence data.
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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.