|
|
| Line 122: |
Line 122: |
| | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseOne" aria-expanded="false" aria-controls="collapseOne"> | | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseOne" aria-expanded="false" aria-controls="collapseOne"> |
| | Laccase TVEL5 in RFC25 BBa_K1615067 | | Laccase TVEL5 in RFC25 BBa_K1615067 |
| − | </a>
| + | https://2015.igem.org/Team:Edinburgh/Improved_Part |
| − | </h4>
| + | |
| − | </div>
| + | |
| − | <div id="collapseOne" class="panel-collapse collapse in" role="tabpanel" aria-labelledby="headingOne">
| + | |
| − | <div class="panel-body"> | + | |
| − | <div class="col-md-6">
| + | |
| − | <p style="color: black;">
| + | |
| − | <h2>Materials</h2>
| + | |
| − | Laccases are glycosylated polyphenol oxidases and have four copper ions per molecule<sup>1</sup>. This allows them to catalyse the reduction of O<sub>2</sub> to 2H<sub>2</sub> while oxidising an aromatic substrate<sup>2</sup>. This laccase is coded by the structural gene <i>lcc</i> in <i>Trametes versicolor</i>, a species of white rot fungus<sup>3</sup>.
| + | |
| − | <br>
| + | |
| − | <br>
| + | |
| − | To increase the functions of laccase, we took the sequence from iGEM12_Bielefeld-Germany laccase BBa_K863030 and codon optimised it for the chassis <i>Esherichia coli</i>. We then made it RFC25 compatible by added the prefix and suffix and removing all illegal restriction sites. This laccase can now be fused to other protein genes with an in-frame 6-nucleotide scar.
| + | |
| − | <br>
| + | |
| − | <br>
| + | |
| − | <sup>1</sup>Lontie, R. (1984). <i>Copper proteins and copper enzymes</i> (Vol. 2). CRC.
| + | |
| − | <br><sup>2</sup>Thurston, C. F. (1994). The structure and function of fungal laccases. <i>Microbiology</i>, 140(1), 19-26.
| + | |
| − | <br><sup>3</sup>Collins, P. J., & Dobson, A. (1997). Regulation of laccase gene transcription in Trametes versicolor. <i>Applied and Environmental Microbiology</i>, 63(9), 3444-3450.
| + | |
| − | | + | |
| − | | + | |
| − | | + | |
| − | </ul>
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | <div class="col-md-6">
| + | |
| − | <p class="text-muted">
| + | |
| − | <h2>Procedure</h2>
| + | |
| − | <ul>
| + | |
| − | <li>1. Mix the agarose with the 1X TAE buffer in a flask.
| + | |
| − | <li>2. Heat the mixture until all the agarose is dissolved.
| + | |
| − | <li>3. Swirl the flask under cold running water to cool the mixture.
| + | |
| − | <li> 4. Add the gel stain.
| + | |
| − | <li>5. Pour into an assembled gel tray and let it cool.
| + | |
| − | </uL>
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <div class="panel panel-default">
| + | |
| − | <div class="panel-heading" role="tab" id="headingTwo">
| + | |
| − | <h4 class="panel-title">
| + | |
| − | <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseTwo" aria-expanded="false" aria-controls="collapseTwo">
| + | |
| − | mRFP (BBa_K1615089)
| + | |
| − | </a>
| + | |
| − | </h4>
| + | |
| − | </div>
| + | |
| − | <div id="collapseTwo" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingTwo">
| + | |
| − | <div class="panel-body">
| + | |
| − | <div class="col-md-6">
| + | |
| − | <p>
| + | |
| − | <h2>Materials</h2>
| + | |
| − | <ul>
| + | |
| − | RFP is important in synthetic biology as a visualisation tool. While multiple versions of RFP exist in the registry, only one is RFC25 compatible. This RFP (BBa_K1351021) contains a Shine-Dalgarno sequence in the RFC25 prefix which precludes it from using the common lac expression cassette (BBa_K314103) which already contains a ribosome binding site and only produces RFC10 fusions. By adding the RFC25 prefix without the Shine-Dalgarno sequence we hope to improve the utility of this RFP.
| + | |
| − | | + | |
| − | We visualised RFP using multiple methods to show that it was folding and expressing.
| + | |
| − | | + | |
| − | <br>
| + | |
| − | 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.
| + | |
| − | <br>
| + | |
| − | <br>
| + | |
| − | 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>
| + | |
| − | <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.
| + | |
| − | | + | |
| − | </ul>
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | <div class="col-md-6">
| + | |
| − | <p>
| + | |
| − | <h2>Procedure</h2>
| + | |
| − | <ul>
| + | |
| − | <li>1. Place gel tray into the electrophoresis apparatus.
| + | |
| − | <li>2. Pour 1X TAE so that the gel is covered by buffer.
| + | |
| − | <li>3. Prepare the samples by adding the appropriate amount of loading dye.
| + | |
| − | <li>4. Load samples and DNA ladder into wells on the gel.
| + | |
| − | <li>5. Run the gel at roughly 100V for around an hour
| + | |
| − | | + | |
| − | </uL>
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <div class="panel panel-default">
| + | |
| − | <div class="panel-heading" role="tab" id="headingThree">
| + | |
| − | <h4 class="panel-title">
| + | |
| − | <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseThree" aria-expanded="false" aria-controls="collapseThree">
| + | |
| − | Monoamine oxidase A BBa_K1615022
| + | |
| − | </a>
| + | |
| − | </h4>
| + | |
| − | </div>
| + | |
| − | <div id="collapseThree" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingThree">
| + | |
| − | <div class="panel-body">
| + | |
| − | <div class="col-md-6">
| + | |
| − | <p>
| + | |
| − | <h2>Materials</h2>
| + | |
| − | <ul>
| + | |
| − | 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>.
| + | |
| − | <br>
| + | |
| − | <br>
| + | |
| − | 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.
| + | |
| − | <br>
| + | |
| − | <br>
| + | |
| − | 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.
| + | |
| − | | + | |
| − | </ul>
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | <div class="col-md-6">
| + | |
| − | <p>
| + | |
| − | <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>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − |
| + | |
| − | <footer>
| + | |
| − | <p class="pull-right"><a href="#">Back to top</a></p>
| + | |
| − | <p>© 2015 EdiGEM · <a href="#">Privacy</a> · <a href="#">Terms</a></p>
| + | |
| − | </footer>
| + | |
| − | | + | |
| − | </body>
| + | |
| − | </html>
| + | |
