Team:Valencia UPV/Project/modules/methodology/luciferase

From 2014.igem.org

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<div align="center"><span class="coda"><roja>L</roja>uciferase <roja>E</roja>xpression <roja>A</roja>ssay</div><br/><br/>
<div align="center"><span class="coda"><roja>L</roja>uciferase <roja>E</roja>xpression <roja>A</roja>ssay</div><br/><br/>
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<p>The implementation of a genetic switch was key in the development of the Sexy Plant. Therefore, we needed an accurate expression analysis technique to test its performance.</p></br/>
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<p>The implementation of a genetic switch was key in the development of the Sexy Plant. Therefore, we needed an accurate expression analysis technique to test its induction capability.</p></br/>
<p>Remember that we introduced an <span class="blue-bold">Inducible genetic switch</span> with the objective of activating the pheromone production upon the insect mating season. The switch is activated with the addition of Copper sulphate to the plant leaves.</p></br/>
<p>Remember that we introduced an <span class="blue-bold">Inducible genetic switch</span> with the objective of activating the pheromone production upon the insect mating season. The switch is activated with the addition of Copper sulphate to the plant leaves.</p></br/>
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<div align="center"><img width="400px" style="margin-right: 30px;" src="http://2014.igem.org/wiki/images/c/c0/VUPV397px-Firefly_composite.png" alt="Bioluminescent_firefly" title="Bioluminescent firefly"></img><img width="250px" style="margin-left: 30px;" src="http://2014.igem.org/wiki/images/6/6d/VUPVRenilla.jpg" alt="sea_pansy" title="Sea Pansy"></img><br/>
<div align="center"><img width="400px" style="margin-right: 30px;" src="http://2014.igem.org/wiki/images/c/c0/VUPV397px-Firefly_composite.png" alt="Bioluminescent_firefly" title="Bioluminescent firefly"></img><img width="250px" style="margin-left: 30px;" src="http://2014.igem.org/wiki/images/6/6d/VUPVRenilla.jpg" alt="sea_pansy" title="Sea Pansy"></img><br/>
<p style="text-align: justify; font-style: italic; font-size: 0.8em; width: 700px;"><span class="black-bold">Figure 1</span>. Bioluminescent firefly
<p style="text-align: justify; font-style: italic; font-size: 0.8em; width: 700px;"><span class="black-bold">Figure 1</span>. Bioluminescent firefly
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Source: Emmanuelm at en.wikipedia.</p>
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Source: <a class="normal-link-page" href="http://en.wikipedia.org/wiki/User:Emmanuelm">Emmanuelm</a> at <a class="normal-link-page" href="http://en.wikipedia.org/wiki/Main_Page">en.wikipedia</a>.</p>
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<p><br/><br/>Therefore, we collected leaf samples from transformed <i>N. benthamiana</i> with our <span class="blue-bold">construct</span>, which was comprised of the switch, the Firefly luciferase reporter, and the <i>Renilla</i> luciferase internal standard. We cut small discs from the leaves and introduced them in a multiwall plate, containing CuSO4 dilutions at different concentrations.</p></br/>
<p><br/><br/>Therefore, we collected leaf samples from transformed <i>N. benthamiana</i> with our <span class="blue-bold">construct</span>, which was comprised of the switch, the Firefly luciferase reporter, and the <i>Renilla</i> luciferase internal standard. We cut small discs from the leaves and introduced them in a multiwall plate, containing CuSO4 dilutions at different concentrations.</p></br/>
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<p>Then the reagent containing the necessary substrates to start the Firefly luciferase reaction (Dual-Glo® LuciferaseAssay Reagent is added and the luminescence values are detected in a GloMax 96 Microplate Luminometer (Promega) and recorded.</p></br/>
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<p>After preparing leaf extracts, the reagent containing the necessary substrate to start the Firefly luciferase reaction (Dual-Glo® Luciferase Assay Reagent) is added and the luminescence values are detected in a GloMax 96 Microplate Luminometer (Promega) and recorded.</p></br/>
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<p>Afterwards, another reagent (Dual-Glo® Stop & Glo® Reagent) is added which can quench the Firefly luciferase luminescence and activate the <i>Renilla</i> luciferase reaction. Then, <i>Renilla</i> luminescence is also detected in the Luminometer and the values are recorded.</p></br/>
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<p>Afterwards, a second reagent (Dual-Glo® Stop & Glo® Reagent) that quenches the Firefly luciferase luminescence and activates the <i>Renilla</i> luciferase reaction is added. Then, <i>Renilla</i> luminescence is also detected in the Luminometer and the values are recorded.</p></br/>
<p>Finally, once both values are recorded for each well, the ratio Firefly/<i>Renilla</i> luminescence is calculated. Since Renilla luminescence is supposed to be constant between samples, this ratio, as mentioned before, allows knowing whether the changes in firefly luminescence are due to changes in expression or external factors. In addition, samples must be normalized with a negative control.</p></br/>
<p>Finally, once both values are recorded for each well, the ratio Firefly/<i>Renilla</i> luminescence is calculated. Since Renilla luminescence is supposed to be constant between samples, this ratio, as mentioned before, allows knowing whether the changes in firefly luminescence are due to changes in expression or external factors. In addition, samples must be normalized with a negative control.</p></br/>
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<p>We also wanted to compare the expression of this chimeric promoter and terminator (<a class="normal-link-page" href="#">see Constructs: Switch</a>) with other well-known promoters and terminators. Therefore, using the following equation, the relative expression ratio or experimental transcriptional activity (ETA) compared to the known expression values of the PNos promoter and the TNos terminator can be calculated. The ETA of PNos and TNos is arbitrarily set as 1.</p><br/>
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<p>We also wanted to compare the expression of this chimeric promoter and terminator (<a class="normal-link-page" href="http://2014.igem.org/Team:Valencia_UPV/Project/results/constructs#switch">see Constructs: Switch</a>) with other well-known promoters and terminators. Therefore, using the following equation, the relative expression ratio or experimental transcriptional activity (ETA) compared to the known expression values of the PNos promoter and the TNos terminator can be calculated. The ETA of PNos and TNos is arbitrarily set as 1.</p><br/>
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<p><span class="red-bold">FALTA METER UNA ECUACION AQUI!!</span></p><br/>
 
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<p>With all this setup, we are ready to know if our Copper inducible switch is really working and quantify its induction of the Luciferase reporter gene expression(<a class="normal-link-page" href="http://2014.igem.org/Team:Valencia_UPV/Project/results">See Results: Luciferase assay</a>).</p><br/>
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<p>With all this setup, we are ready to know if our Copper inducible switch is really working and quantify its induction of the Luciferase reporter gene expression.</p><br/>
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<p>We obtained preliminary expression results that showed a weak induction of the Firefly luciferase in the leaf discs. Discs were sprayed with 100µM CuSO4 and the Firefly Luciferase and <i>Renilla</i> Luciferase activities measured after 12h. Copper treated discs showed FLuc/RLuc values 1,3 times higher than the control with an ETA of 0,14 meaning that this switch induction under these conditions is lower than the PNos-Tnos expression. Therefore, further studies with different conditions and concentrations are required.</p><br/><br/><br/>

Revision as of 00:58, 18 October 2014

Project > Modules > Methodology > Luciferase expression assay



Luciferase Expression Assay


The implementation of a genetic switch was key in the development of the Sexy Plant. Therefore, we needed an accurate expression analysis technique to test its induction capability.


Remember that we introduced an Inducible genetic switch with the objective of activating the pheromone production upon the insect mating season. The switch is activated with the addition of Copper sulphate to the plant leaves.


In order to test the expression induced by the switch, we decided to perform a Luciferase assay (Dual-Glo ® Luciferase Assay system, Promega). This assay is based on the use of two luminescent proteins, one from an insect, the Firefly Luciferase and the other one from the Sea pansy, the Renilla luciferase. The first one is used as a reporter, being expressed when the switch is activated, and the other one is used as an internal standard, which allows differentiating if changes in the expression of the reporter are due to the switch induction or other factors.


Bioluminescent_fireflysea_pansy

Figure 1. Bioluminescent firefly Source: Emmanuelm at en.wikipedia.


Figure 2. Sea pansy, Renilla reniformis Source: http://www.jaxshells.org/



Therefore, we collected leaf samples from transformed N. benthamiana with our construct, which was comprised of the switch, the Firefly luciferase reporter, and the Renilla luciferase internal standard. We cut small discs from the leaves and introduced them in a multiwall plate, containing CuSO4 dilutions at different concentrations.


After preparing leaf extracts, the reagent containing the necessary substrate to start the Firefly luciferase reaction (Dual-Glo® Luciferase Assay Reagent) is added and the luminescence values are detected in a GloMax 96 Microplate Luminometer (Promega) and recorded.


Afterwards, a second reagent (Dual-Glo® Stop & Glo® Reagent) that quenches the Firefly luciferase luminescence and activates the Renilla luciferase reaction is added. Then, Renilla luminescence is also detected in the Luminometer and the values are recorded.


Finally, once both values are recorded for each well, the ratio Firefly/Renilla luminescence is calculated. Since Renilla luminescence is supposed to be constant between samples, this ratio, as mentioned before, allows knowing whether the changes in firefly luminescence are due to changes in expression or external factors. In addition, samples must be normalized with a negative control.


luciferase

Figure 3. Dual-Glo ® Luciferase Assay system procedure Source: Dual-Glo ® Luciferase Assay system Technical Manual (Promega) .


We also wanted to compare the expression of this chimeric promoter and terminator (see Constructs: Switch) with other well-known promoters and terminators. Therefore, using the following equation, the relative expression ratio or experimental transcriptional activity (ETA) compared to the known expression values of the PNos promoter and the TNos terminator can be calculated. The ETA of PNos and TNos is arbitrarily set as 1.



With all this setup, we are ready to know if our Copper inducible switch is really working and quantify its induction of the Luciferase reporter gene expression.


We obtained preliminary expression results that showed a weak induction of the Firefly luciferase in the leaf discs. Discs were sprayed with 100µM CuSO4 and the Firefly Luciferase and Renilla Luciferase activities measured after 12h. Copper treated discs showed FLuc/RLuc values 1,3 times higher than the control with an ETA of 0,14 meaning that this switch induction under these conditions is lower than the PNos-Tnos expression. Therefore, further studies with different conditions and concentrations are required.




← Go back to Methodology Go to Sample Analysis →


REFERENCES


  1. Dual-Glo ® Luciferase Assay system Technical Manual (Promega).
  2. Sarrion-Perdigones A, Vazquez-Vilar M, Palací J, Castelijns B, Forment J, Ziarsolo P, Blanca J, Granell A, Orzaez D. (2013) GoldenBraid2.0: A comprehensive DNA assembly framework for Plant Synthetic Biology. Plant Physiol Epub ahead of print, doi: 10. 1104/ pp. 113. 217661.