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>
 
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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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                   <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>   
 
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         <div class="panel-heading" role="tab" id="headingOne">
 
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               Heroin Esterase BBa_K1615045
 
               Heroin Esterase BBa_K1615045
 
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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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              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<sup>1</sup>. The gene <i>her</i> encodes this enzyme and has the ability to be expressed in the chassis Escherichia coli<sup>2</sup>. The pH optimum for this enzyme to function is in 8.5 in bicine buffer<sup>1</sup>.
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<br>
 
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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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The activity of heroin esterase can be tested using 4-nitrophenyl acetate which is hydrolised(?) by heroin esterase to form 4-nitrophenol + actetate. This produces a yellow colour as well as being able to be read at 410 nm.
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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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<br>
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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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<br>
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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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Design: The sequence for our enzyme used the original sequence from Rathbone, et al., and was then codon optimised for <i>E. coli</i>. The RFC25 prefix and suffix were added along 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.
 
 
<br>
 
<br>
 
<br>
 
<br>
 
<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.  
 
<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><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>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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              <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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              <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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              <ul>
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                The structural gene morphine-6-dehyrogenase (<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 catalyses the oxidation of both morphine and codeine to produce morphinone and codeinone. During this process NADP<sup>+</sup> is reduced to NADPH which means that it is frequently used to detect morphine and codeine enzymatically<sup>2</sup>.
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To test the morphine dehydrogenase activity it can be coupled with codeine and NADP<sup>+</sup> to produce codeinone and NADPH. The amount of NADPH produced can be measured at x nm.
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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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<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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<img src="https://static.igem.org/mediawiki/2015/2/2f/Morpg.jpg" class="img-responsive">
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<br>Following table summarises the kinetic analysis and statistics of the measurment.
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<img src="https://static.igem.org/mediawiki/2015/e/e1/Morphgraph.jpg" class="img-responsive">
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<br>
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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.
 
<br>
 
<br>
 
<br>
 
<br>
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Design: To make this gene standardised it was codon optomised for the chassis <i>Esherichia coli</i> as well as making it RFC25 compatible which required getting rid of all illegal restriction sites in the gene sequence.
 
 
<br>
 
<br>
 
<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.
 
<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>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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              <h2>Procedure</h2>
 
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                <li>1. Place gel tray into the electrophoresis apparatus.
 
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                <li>2. Pour 1X TAE so that the gel is covered by buffer.
 
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                <li>4. Load samples and DNA ladder into wells on the gel.
 
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                <li>5. Run the gel at roughly 100V for around an hour
 
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            <div align="center">
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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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            </div>
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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>.  
 
                 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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<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.
 
<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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            <div class="col-md-6">
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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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              <p>
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            </div>
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              <h2>Procedure</h2>
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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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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.
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