http://2012.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=100&target=MarkWeijers&year=&month=2012.igem.org - User contributions [en]2024-03-28T23:37:10ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Team:TU-Delft/part1Team:TU-Delft/part12012-12-17T13:05:25Z<p>MarkWeijers: </p>
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<head><title>Receptor</title></head><br />
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<h2>Content</h2> <br />
<a href="#P10"> Introduction</a><br><br />
<a href="#P8">Parts </a><br><br />
<a href="#P7">Results </a><br><br />
<a href="#P6"> Conclusions</a><br><br />
<a href="#P5"> Recommendations </a><br><br />
<a href="#P9">References</a><br><br />
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<a name="P10"> <br><h2>Introduction</h2> </a><br />
<h3>Olfactory receptors</h3> <br />
<p><br />
Animals sense their chemical environment through olfactory receptors (ORs). The olfactory receptors are a large group of proteins belonging to a subfamily of G protein-coupled receptors (GPCRs) that bind odorant ligands. If the receptor is activated by a ligand, the confirmation of the receptor is changed and there is an interaction with the a-subunit of the G-protein. This causes dissociation of the a-subunit from the Gßα dimer and the signal is propagated [1]. Because of the sensitivity and selectivity of the of the olfactory system it can be of value in detection of environmental toxins [2] or pharmaceutical screening. In this iGEM project we aim to investigate if the ORs can be used as a diagnostics tool for tuberculosis.</p><br />
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<h3>Yeast G protein-coupled receptors</h3><a name="A4"> </a> <br />
<p>In this project we choose to work with the budding yeast <i>Saccharomyces cerevisiae</i> as a host organism because it utilizes already a GPCR pathway. Furthermore <i>S. cerevisiae</i> has been successfully used for functional expression of GPCR’s [3,4], a lot of genomic tools are available, and it has a fully characterized genome. <br />
In <i>S. cerevisiae</i> two GPCR cascades have been identified: a glucose sensing pathway and a mating pathway [5]. There are two sexes of yeast cells, MATa and MATa. Whenever pheromones (small peptides) of the opposite sex are bound to the specific G-protein coupled receptors (Ste2 p or Ste3p), the MAP kinase cascade is turned on, leading to induction of mating genes such as <i>FUS1</i> and growth arrest mediated by the <i>FAR1</i> promoter. This mating response can be seen in the form of a morphological change, called shmoo formation. In figure 1 an overview of the pheromone and glucose signaling pathways in <i>S. cerevisiae</i> is shown. </p><br />
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<img src="https://static.igem.org/mediawiki/igem.org/4/4a/GPCRinyeast.png" height="425" width="425" /><br />
<h6>Overview of pheromone and glucose signaling in <i>S. cerevisiae</i>. Figure adapted from <i>Versele et al.</i></h6><br />
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<h3>Introduction of a new olfactory receptor</h3> <a name="A3"> </a><br />
<p>Previously it was found that that the yeast pheromone signaling pathway can be coupled to a mammalian olfactory receptor. <i> Minic et al.</i> succeeded in functional expressing the rat 17 OR and its trafficking to the plasma membrane in <i>S. cerevisiae</i>. Between the three GPCRs that are known in <i>S. cerevisiae</i>, Ste2, Ste3 and Gpr1, the sequence similarity is limited. Except for pheromone receptors in <i>Schizosaccharomyces pombe</i> and <i>Kluyveromyces lactis</i>, Ste2 and Ste3 are largely unrelated in sequence to other GPCRs [5]. Nevertheless, the yeast pheromone receptors can be functionally replaced by several mammalian GPCRs so that the pheromone pathway can be activated by the corresponding ligands [4]. For localization of the receptor into the membrane and a higher affinity with the alpha-subunit we made a chimeric design of the receptor, after the research of Radhika et al. [7]. For a detailed design, and how to do this yourself, look at the <a href="https://2012.igem.org/Team:TU-Delft/receptordesign">DIY receptor design page</a>.</p><br />
<h3>Niacin olfactory receptor</h3> <a name="A5"> </a> <br />
<p>The receptors GPR109A and GPR109B are known to bind the compound nicotinic acid [8]. It was previously described that GPR109B acts a low affinity receptor for nicotinic acid and GPR109A acts as a high affinity receptor for nicotinic acid and other compounds with related pharmacology [molecular identification of high and low affinity receptors]. The chemical compound methyl nicotinate is closely related to nicotinic acid. Because one of the compounds in the breath of tuberculosis patients is methyl nicotinate [9,10], the high affinity receptor for niacin is a good candidate for testing the ‘olfactory yeast’ as a diagnostics tool. </p><br />
<h3>Banana smell olfactory receptor</h3><a name="B1"> </a><br />
<p>The first iGEM team of MIT 2006 made a biobrick called the ‘banana odor generator’. With this part <i>E. coli</i> cells can generate the isoamyl acetate molecule. We aim to let yeast detect this isoamyl acetate signal with a olfactory receptor. The idea is that in future work the yeast should couple this back to the bacteria to have gaseous yeast/bacteria communication on plates. <br/> The human receptor OR1G1 and mouse receptor Olfr154 are known to react on isoamyl acetate [11] and therefor these two receptors were used in this iGEM project. </p><br/><br />
<a name="P8"><h2>Parts</h2> </a><br />
<p>A design of the receptor construct was made with the olfactory receptors placed between the N-terminal and the C-terminal part of the rat I7 receptor. As a promoter the strong constitutive <i>GPD</i> promoter is used and as a terminator the <i>CYC1</i> terminator. The receptor can be replaced by using the restriction sites BamHI and NdeI. A FLAG tag is added upstream of the receptor sequence to look at the localization of the receptor in the membrane.<br />
The plasmid construct for the receptor expression was obtained by restriction of the synthesized receptor construct and ligation in the pRSII415 expression vector. The following biobricks are created:<br/><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775000 " target="_blank">BBa_K775000</a><br/><br />
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<img src="https://static.igem.org/mediawiki/igem.org/b/b9/GPR109A_picture.jpg" width="425" /><br />
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<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775001 " target="_blank">BBa_K775001</a><br/><br />
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<img src="https://static.igem.org/mediawiki/igem.org/8/8d/Olfr154_picture.jpg" width="425" /><br />
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<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775002 " target="_blank">BBa_K775002</a><br/><br />
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<img src="https://static.igem.org/mediawiki/igem.org/9/9f/OR1G1_picture.jpg" width="425" /><br />
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<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775003 " target="_blank">BBa_K775003</a><br/></p><br />
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<img src="https://static.igem.org/mediawiki/igem.org/c/c7/Odr10_picture.jpg" width="425" /><br />
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<a name="P7"><h2>Results</h2></a><br />
<h3>Transformations</h3><br />
The transformations with the plasmids were done in the S288C <i>S. cerevisiae</i> strain. All the plasmids were also put in a strain with a far1::kanMX4 knock-out strain. In table 1 all the strains that we made in this subpart are shown.<br />
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<center><br />
<table id="tbtext" width=100% ><br />
<tr> <br />
<th></th> <th>Gpr109A</th> <th>Olfr154</th> <th>OR1G1</th></tr><br />
<tr><td id="tdunderline">Normal strain</td><td id="tdunderline">+NR1</td><td id="tdunderline">+BR1</td><td id="tdunderline">+BR2</td></tr><br />
<tr><td id="tdunderline">&Delta;far1 strain</td><td id="tdunderline">&Delta;far1 +NR1</td><td id="tdunderline">&Delta;far1 +BR1</td><td id="tdunderline">&Delta;far1 +BR2</td></tr><br />
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</table><br />
</center><br />
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<p>After transformation of the plasmids in the yeast a PCR reaction was performed in order to verify if the plasmid was correctly transformed. Since the PCR reactions were performed with single colonies we expected to obtain one PCR product with the length of the receptor part. However, for all the receptors we saw multiple PCR products on the gel; products with the length of the receptor, and products indicating that only the plasmid backbone was present (without the receptor). This indicates that during growth of the yeast a part of the plasmid was emitted. </p><br />
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<img src="https://static.igem.org/mediawiki/2012/0/0a/DNA_gel_typical_Receptor.png" height="30%" width="30%" /><br />
<h6>1% Agarose Gel run on 80 V, showing <i>S. cerevisiae</i> extracted plasmid DNA of our olfactory receptor construct. Lane 2 shows DNA smartladder. Lane 1 shows a typical bands for the <i>S. cerevisae</i> plasmid extract. The bright band at the height of 2000 nt is the expected PCR band. The secondary bands observed have DNA sizes of approximately 1200 and 400-500 nt.</h6><br />
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<h3>Expression and localization of the ORs</h3><br />
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<a id="FLAG_setup"><h6>Setup</h6></a><br />
NR1, BR1 and BR2 transformed cells and WT cells were stained with conjugated anti-FLAG antibodies according to <a href="https://2012.igem.org/Team:TU-Delft/protocols#P11">the immunofluorescence staining protocol</a> and viewed under a widefield fluorescence microscope (NR1) and a confocal microscope (BR1 and BR2) with the goal of imaging the expression of our GPCR chimeras and image their localization in the cell. The image was analyzed with ImageJ to compare the fluorescence of the cell and cell membrane to the overall fluorescence of the whole picture.<br />
<a id="FLAG_outcome"><h6>Outcome</h6></a><br />
It can be seen that there is expression of the receptor: +NR1 cells are fluorescent (figure 4 below) and the reference strain is very weakly fluorescent (figure 4 top). In some of the +NR1 cells there is clear halo structure visible, which indicates localization of the receptor on the membrane. Below such a typical halo is shown (figure 4). For +BR1 and +BR2 cells receptor expression can be shown also by the fluorescent signal (figure 5, other experiment). The location of the signal indicates expression unequally distributed over the cells, indicating that localization in the membrane is not fully functioning. Contrast of these pictures are not optimal. The reference strain (figure 5, top) showed very little fluorescence. Further, a small percentage of the cells (~5 %) was found to be fluorescent, indicating that constitutive mRNA expression by the GPD promoter did not lead to constitutive protein expression in all cells.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2012/1/15/NR1_immuno.png" alt="some_text" width="300" align="center"><br />
<p><br />
<h6>Figure 4: FLAG immunolocalization of +NR1 yeast cells expressing a receptor with DYKDDDDK tag.</h6><br />
</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/08/Banana_receptor_summary.png" alt="some_text" width="450" ><br />
<p> <br />
<h6>Figure 5: FLAG immunolocalization of +BR1 and +BR2 yeast cells expressing a receptor with FLAGtag.</h6><br />
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<h3>Ligand activation</h3><br />
<h6>Setup</h6><br />
<p>The next step was to test the binding of the ligand to our new introduced receptor. If the ligand binds correctly to the receptor the downstream pathway is activated and the cells should go in growth arrest. If cells are dividing the percentage of DNA is higher then for cells that are in growth arrest. By staining the DNA of the cells with a fluorescent dye we could look at the DNA content and thus if the cells enter to growth arrest. This is done with a flow cytometer.<br />
<h6>Outcome <i>Niacin receptor</i></h6><br><br />
<img src="https://static.igem.org/mediawiki/igem.org/0/0d/DNAstainingNR1.jpg" align="middle" width="100%"/><br />
<h6>Flow cytometry results of +NR1 and reference cells induced with the niacin ligand. Cells were DNA stained and measured after 4.40 hours.</h6><br/><br />
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As controls we used a reference strain induced with niacin, and a +NR1 receptor strain without niacin induction. On the right there is our +NR1 receptor strain induced with niacin. The graphs show on the x-axis the measured intensity and on the y-axis we see the cell count. <br><br />
We see that the controls show a peak in the same region, this peak indicates that the cells are distributed over all cell cycle phases. We can see that the peak of the induced receptor strain is shifted to a lower fluorescent intensity. This might be an indication that the cells have a lower DNA content, and that they stopped growing. <br />
Despite we didn’t see a response on the methyl nicotinate molecule we were very exited to have promising results on the binding of the niacin ligand! This is also in agreement with the results of the <a href="https://2012.igem.org/Team:TU-Delft/Modeling/StructuralModeling">molecular dynamics modeling</a>.<br />
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<h6><i>Outcome Banana receptor 1 and Banana receptor 2</i></h6><br><br />
When looking at the stained +BR1 and +BR2 strains with flow cytometry the data showed unclear and strangely shifted peaks. To get an idea of the behavior of the cell under influence of ligand and DNA staining, the cells were viewed with fluorescence microscopy. There was an effect of isoamylacetate on the location of the DNA stain. For all the strains this resulted in an evenly distributed glow over the whole cell after induction of isoamylacetate. Below microscopy pictures for the +BR1 strain are shown as an example. This observations could explain the unclear flow cytometry results. Another reason for that could be that the oily isoamyl acetate that drove on the medium disturbed the measurements. <br />
</p><br />
<img src="https://static.igem.org/mediawiki/2012/5/51/Tud_Image002e.jpg" width="400" alt="boebeloeba"/><br />
<p><br />
<h6>Figure 7: OR1G1 transformed cells with isoamylacetate as inducing agent at an estimated concentration of 200mM.</h6><br />
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<a name="P6"><h2>Conclusions</h2> </a><br />
All the olfactory receptors were successfully expressed in the yeast strains. For some of the receptors we observed a halo structure with FLAG-tag localization, this points to localization of the receptor into the membrane. <br><br />
When inducing the niacin receptor strain with niacin we observed that the flow cytometry peak of the induced receptor strain was shifted to a lower fluorescent intensity. This might be an indication that the cells stopped growing and thus there was a reaction on the ligand! For the banana receptor strains we had difficulties with the DNA staining in combination with the ligand Isoamyl acetate. <br />
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<a name="P5"><h2>Recommendations</h2> </a><br />
<p>During growth of yeast cells transformed with the expression vector we observed two things: not all the cells maintain the right plasmid and the cells grew slower than wild type cells. A reason for this could be that the expression of the receptor is disadvantageously for the cells. Therefor we recommend for future work to use an inducible promoter instead of a strong constitutive promoter. In that case one can make the yeast cells expressing the receptor just before testing the strain.</p><br/><br />
<a name="P9"><h2>References</h2> </a><br />
<h6><br />
[1] Haiqing Zhao, Lidija Ivic, Joji M. Otaki, Mitsuhiro Hashimoto, Katsuhiro Mikoshiba, Stuart Firestein*Functional Expression of a Mammalian Odorant Receptor, Science 279, 237 (1998)<br/><br />
[2] Venkat Radhika, Tassula Proikas-Cezanne, Muralidharan Jayaraman, Djamila Onesime, Ji Hee Ha &Danny N Dhanasekaran, Chemical sensing of DNT by engineered olfactory yeast strain Nature Chemical Biology 3 (2007) <br/><br />
[3] Jasmina Minic, Marie-annick Persuy, Elodie Godel, Josiane Aioun, Ian Connerton, Roland Salesse, Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening, FEBS Journal (2005)<br/><br />
[4] Brown et al, Functional coupling of mammalian receptors to the yeast mating pathway using novel yeast/mammalian G protein a-subunit chimeras, Yeast (2000)<br/><br />
[5] Matthias Versele, Katleen Lemaire, and Johan M. Thevelein, Sex and sugar in yeast: two distinct GPCR systems, EMBO Rep. 2001<br/><br />
[6] Dietmar Krautwurst, King-Wai Yau, and Randall R. Reed, Identification of Ligands for Olfactory Receptors by Functional Expression of a Receptor Library, Cell (1998)<br/><br />
[7] Venkat Radhika, Tassula Proikas-Cezanne, Muralidharan Jayaraman, Djamila Onesime, Ji Hee Ha & Danny N Dhanasekaran, Chemical sensing of DNT by engineered olfactory yeast strain, Nature Chemical biology (2007)<br/><br />
[8] Alan Wise, Steven M. Foord, Neil J. Fraser, Ashley A. Barnes,e Nabil Elshourbagy, Michelle Eilert,g Diane M. Ignarg Paul R. Murdock, Klaudia Steplewski,h Andrew Green,Andrew J. Brown, Simon J. Dowell, Philip G. Szekeres, David G. Hassall, Fiona H. Marshall,a, j Shelagh Wilson, and Nicholas B. Pike<br />
Molecular Identification of High and Low Affinity Receptors for Nicotinic Acid, The journal of biological chemistry (2003) <br/><br />
[9] Georgies F. Mgode Bart J. Weetjens Thorben Nawrath, Christophe Cox, Maureen Jubitana, Robert S. Machang’, Stephan Cohen-Bacrie,e Marielle Bedotto, Michel Drancourt,e Stefan Schulz and Stefan H. E. Kaufmann, Diagnosis of Tuberculosis by Trained African Giant Pouched Rats and Confounding Impact of Pathogens and Microflora of the Respiratory Tract, Journal of clinical microbiology (2011) <br/><br />
[10] Mona Syhre, Stephen T. Chambers, The scent of Mycobacterium tuberculosis, Elsevier (2008)<br/><br />
[11 Valery Matarazzo, Olivier Clot-Faybesse, Brice Marcet, Gaelle Guiraudie-Capraz, Boriana Atanasova, Gerard Devauchelle, Martine Cerutti, Patrick Etievant and Catherine Ronin,Functional Characterization of Two Human Olfactory Receptors Expressed in the Baculovirus Sf9 Insect Cell System, Chem. Senses (2005).</h6><br/><br />
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<a href='https://2012.igem.org/Main_Page' target="_blank"><div id='logo_igem2'></div><a/></body></html></div>MarkWeijershttp://2012.igem.org/Team:TU-Delft/part1Team:TU-Delft/part12012-12-17T13:02:58Z<p>MarkWeijers: </p>
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<head><title>Receptor</title></head><br />
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<div id="logo_ed"><a href="https://2012.igem.org/Team:TU-Delft" 'onfocus=this.blur()'><img src="https://static.igem.org/mediawiki/igem.org/8/88/Logoigemklein.png" border="0" width="100" height="100"/></a></div><br />
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<h2>Content</h2> <br />
<a href="#P10"> Introduction</a><br><br />
<a href="#P8">Parts </a><br><br />
<a href="#P7">Results </a><br><br />
<a href="#P6"> Conclusions</a><br><br />
<a href="#P5"> Recommendations </a><br><br />
<a href="#P9">References</a><br><br />
<br />
<a name="P10"> <br><h2>Introduction</h2> </a><br />
<h3>Olfactory receptors</h3> <br />
<p><br />
Animals sense their chemical environment through olfactory receptors (ORs). The olfactory receptors are a large group of proteins belonging to a subfamily of G protein-coupled receptors (GPCRs) that bind odorant ligands. If the receptor is activated by a ligand, the confirmation of the receptor is changed and there is an interaction with the a-subunit of the G-protein. This causes dissociation of the a-subunit from the Gßα dimer and the signal is propagated [1]. Because of the sensitivity and selectivity of the of the olfactory system it can be of value in detection of environmental toxins [2] or pharmaceutical screening. In this iGEM project we aim to investigate if the ORs can be used as a diagnostics tool for tuberculosis.</p><br />
<br />
<h3>Yeast G protein-coupled receptors</h3><a name="A4"> </a> <br />
<p>In this project we choose to work with the budding yeast <i>Saccharomyces cerevisiae</i> as a host organism because it utilizes already a GPCR pathway. Furthermore <i>S. cerevisiae</i> has been successfully used for functional expression of GPCR’s [3,4], a lot of genomic tools are available, and it has a fully characterized genome. <br />
In <i>S. cerevisiae</i> two GPCR cascades have been identified: a glucose sensing pathway and a mating pathway [5]. There are two sexes of yeast cells, MATa and MATa. Whenever pheromones (small peptides) of the opposite sex are bound to the specific G-protein coupled receptors (Ste2 p or Ste3p), the MAP kinase cascade is turned on, leading to induction of mating genes such as <i>FUS1</i> and growth arrest mediated by the <i>FAR1</i> promoter. This mating response can be seen in the form of a morphological change, called shmoo formation. In figure 1 an overview of the pheromone and glucose signaling pathways in <i>S. cerevisiae</i> is shown. </p><br />
<br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/4/4a/GPCRinyeast.png" height="425" width="425" /><br />
<h6>Overview of pheromone and glucose signaling in <i>S. cerevisiae</i>. Figure adapted from <i>Versele et al.</i></h6><br />
<br/><br />
<h3>Introduction of a new olfactory receptor</h3> <a name="A3"> </a><br />
<p>Previously it was found that that the yeast pheromone signaling pathway can be coupled to a mammalian olfactory receptor. <i> Minic et al.</i> succeeded in functional expressing the rat 17 OR and its trafficking to the plasma membrane in <i>S. cerevisiae</i>. Between the three GPCRs that are known in <i>S. cerevisiae</i>, Ste2, Ste3 and Gpr1, the sequence similarity is limited. Except for pheromone receptors in <i>Schizosaccharomyces pombe</i> and <i>Kluyveromyces lactis</i>, Ste2 and Ste3 are largely unrelated in sequence to other GPCRs [5]. Nevertheless, the yeast pheromone receptors can be functionally replaced by several mammalian GPCRs so that the pheromone pathway can be activated by the corresponding ligands [4]. For localization of the receptor into the membrane and a higher affinity with the alpha-subunit we made a chimeric design of the receptor, after the research of Radhika et al. [7]. For a detailed design, and how to do this yourself, look at the <a href="https://2012.igem.org/Team:TU-Delft/receptordesign">DIY receptor design page</a>.</p><br />
<h3>Niacin olfactory receptor</h3> <a name="A5"> </a> <br />
<p>The receptors GPR109A and GPR109B are known to bind the compound nicotinic acid [8]. It was previously described that GPR109B acts a low affinity receptor for nicotinic acid and GPR109A acts as a high affinity receptor for nicotinic acid and other compounds with related pharmacology [molecular identification of high and low affinity receptors]. The chemical compound methyl nicotinate is closely related to nicotinic acid. Because one of the compounds in the breath of tuberculosis patients is methyl nicotinate [9,10], the high affinity receptor for niacin is a good candidate for testing the ‘olfactory yeast’ as a diagnostics tool. </p><br />
<h3>Banana smell olfactory receptor</h3><a name="B1"> </a><br />
<p>The first iGEM team of MIT 2006 made a biobrick called the ‘banana odor generator’. With this part <i>E. coli</i> cells can generate the isoamyl acetate molecule. We aim to let yeast detect this isoamyl acetate signal with a olfactory receptor. The idea is that in future work the yeast should couple this back to the bacteria to have gaseous yeast/bacteria communication on plates. <br/> The human receptor OR1G1 and mouse receptor Olfr154 are known to react on isoamyl acetate [11] and therefor these two receptors were used in this iGEM project. </p><br/><br />
<a name="P8"><h2>Parts</h2> </a><br />
<p>A design of the receptor construct was made with the olfactory receptors placed between the N-terminal and the C-terminal part of the rat I7 receptor. As a promoter the strong constitutive <i>GPD</i> promoter is used and as a terminator the <i>CYC1</i> terminator. The receptor can be replaced by using the restriction sites BamHI and NdeI. A FLAG tag is added upstream of the receptor sequence to look at the localization of the receptor in the membrane.<br />
The plasmid construct for the receptor expression was obtained by restriction of the synthesized receptor construct and ligation in the pRSII415 expression vector. The following biobricks are created:<br/><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775000 " target="_blank">BBa_K775000</a><br/><br />
<br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/b/b9/GPR109A_picture.jpg" width="425" /><br />
<br/><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775001 " target="_blank">BBa_K775001</a><br/><br />
<br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/8d/Olfr154_picture.jpg" width="425" /><br />
<br/><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775002 " target="_blank">BBa_K775002</a><br/><br />
<br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/9/9f/OR1G1_picture.jpg" width="425" /><br />
<br/><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775003 " target="_blank">BBa_K775003</a><br/></p><br />
<br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/c/c7/Odr10_picture.jpg" width="425" /><br />
<br/><br />
<a name="P7"><h2>Results</h2></a><br />
<h3>Transformations</h3><br />
The transformations with the plasmids were done in the S288C <i>S. cerevisiae</i> strain. All the plasmids were also put in a strain with a far1::kanMX4 knock-out strain. In table 1 all the strains that we made in this subpart are shown.<br />
<br/><br />
<center><br />
<table id="tbtext" width=100% ><br />
<tr> <br />
<th></th> <th>Niacin receptor Gpr109A</th> <th>Banana receptor 1 Olfr154</th> <th>Banana receptor 2 OR1G1</th></tr><br />
<tr><td id="tdunderline">Normal strain</td><td id="tdunderline">+NR1</td><td id="tdunderline">+BR1</td><td id="tdunderline">+BR2</td></tr><br />
<tr><td id="tdunderline">&Delta;far1 strain</td><td id="tdunderline">&Delta;far1 +NR1</td><td id="tdunderline">&Delta;far1 +BR1</td><td id="tdunderline">&Delta;far1 +BR2</td></tr><br />
<br />
</table><br />
</center><br />
<br/><br />
<p>After transformation of the plasmids in the yeast a PCR reaction was performed in order to verify if the plasmid was correctly transformed. Since the PCR reactions were performed with single colonies we expected to obtain one PCR product with the length of the receptor part. However, for all the receptors we saw multiple PCR products on the gel; products with the length of the receptor, and products indicating that only the plasmid backbone was present (without the receptor). This indicates that during growth of the yeast a part of the plasmid was emitted. </p><br />
<br />
<img src="https://static.igem.org/mediawiki/2012/0/0a/DNA_gel_typical_Receptor.png" height="30%" width="30%" /><br />
<h6>1% Agarose Gel run on 80 V, showing <i>S. cerevisiae</i> extracted plasmid DNA of our olfactory receptor construct. Lane 2 shows DNA smartladder. Lane 1 shows a typical bands for the <i>S. cerevisae</i> plasmid extract. The bright band at the height of 2000 nt is the expected PCR band. The secondary bands observed have DNA sizes of approximately 1200 and 400-500 nt.</h6><br />
<br><br />
<br />
<h3>Expression and localization of the ORs</h3><br />
<div class=WordSection1><br />
<br />
<a id="FLAG_setup"><h6>Setup</h6></a><br />
NR1, BR1 and BR2 transformed cells and WT cells were stained with conjugated anti-FLAG antibodies according to <a href="https://2012.igem.org/Team:TU-Delft/protocols#P11">the immunofluorescence staining protocol</a> and viewed under a widefield fluorescence microscope (NR1) and a confocal microscope (BR1 and BR2) with the goal of imaging the expression of our GPCR chimeras and image their localization in the cell. The image was analyzed with ImageJ to compare the fluorescence of the cell and cell membrane to the overall fluorescence of the whole picture.<br />
<a id="FLAG_outcome"><h6>Outcome</h6></a><br />
It can be seen that there is expression of the receptor: +NR1 cells are fluorescent (figure 4 below) and the reference strain is very weakly fluorescent (figure 4 top). In some of the +NR1 cells there is clear halo structure visible, which indicates localization of the receptor on the membrane. Below such a typical halo is shown (figure 4). For +BR1 and +BR2 cells receptor expression can be shown also by the fluorescent signal (figure 5, other experiment). The location of the signal indicates expression unequally distributed over the cells, indicating that localization in the membrane is not fully functioning. Contrast of these pictures are not optimal. The reference strain (figure 5, top) showed very little fluorescence. Further, a small percentage of the cells (~5 %) was found to be fluorescent, indicating that constitutive mRNA expression by the GPD promoter did not lead to constitutive protein expression in all cells.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2012/1/15/NR1_immuno.png" alt="some_text" width="300" align="center"><br />
<p><br />
<h6>Figure 4: FLAG immunolocalization of +NR1 yeast cells expressing a receptor with DYKDDDDK tag.</h6><br />
</p><br />
<img src="https://static.igem.org/mediawiki/2012/0/08/Banana_receptor_summary.png" alt="some_text" width="450" ><br />
<p> <br />
<h6>Figure 5: FLAG immunolocalization of +BR1 and +BR2 yeast cells expressing a receptor with FLAGtag.</h6><br />
<br />
<br />
<br />
</div><br />
<br/><br />
<br />
<br />
<br />
<h3>Ligand activation</h3><br />
<h6>Setup</h6><br />
<p>The next step was to test the binding of the ligand to our new introduced receptor. If the ligand binds correctly to the receptor the downstream pathway is activated and the cells should go in growth arrest. If cells are dividing the percentage of DNA is higher then for cells that are in growth arrest. By staining the DNA of the cells with a fluorescent dye we could look at the DNA content and thus if the cells enter to growth arrest. This is done with a flow cytometer.<br />
<h6>Outcome <i>Niacin receptor</i></h6><br><br />
<img src="https://static.igem.org/mediawiki/igem.org/0/0d/DNAstainingNR1.jpg" align="middle" width="100%"/><br />
<h6>Flow cytometry results of +NR1 and reference cells induced with the niacin ligand. Cells were DNA stained and measured after 4.40 hours.</h6><br/><br />
<br />
As controls we used a reference strain induced with niacin, and a +NR1 receptor strain without niacin induction. On the right there is our +NR1 receptor strain induced with niacin. The graphs show on the x-axis the measured intensity and on the y-axis we see the cell count. <br><br />
We see that the controls show a peak in the same region, this peak indicates that the cells are distributed over all cell cycle phases. We can see that the peak of the induced receptor strain is shifted to a lower fluorescent intensity. This might be an indication that the cells have a lower DNA content, and that they stopped growing. <br />
Despite we didn’t see a response on the methyl nicotinate molecule we were very exited to have promising results on the binding of the niacin ligand! This is also in agreement with the results of the <a href="https://2012.igem.org/Team:TU-Delft/Modeling/StructuralModeling">molecular dynamics modeling</a>.<br />
<br />
<br />
<br/><br><br />
<h6><i>Outcome Banana receptor 1 and Banana receptor 2</i></h6><br><br />
When looking at the stained +BR1 and +BR2 strains with flow cytometry the data showed unclear and strangely shifted peaks. To get an idea of the behavior of the cell under influence of ligand and DNA staining, the cells were viewed with fluorescence microscopy. There was an effect of isoamylacetate on the location of the DNA stain. For all the strains this resulted in an evenly distributed glow over the whole cell after induction of isoamylacetate. Below microscopy pictures for the +BR1 strain are shown as an example. This observations could explain the unclear flow cytometry results. Another reason for that could be that the oily isoamyl acetate that drove on the medium disturbed the measurements. <br />
</p><br />
<img src="https://static.igem.org/mediawiki/2012/5/51/Tud_Image002e.jpg" width="400" alt="boebeloeba"/><br />
<p><br />
<h6>Figure 7: OR1G1 transformed cells with isoamylacetate as inducing agent at an estimated concentration of 200mM.</h6><br />
<br />
<br />
<a name="P6"><h2>Conclusions</h2> </a><br />
All the olfactory receptors were successfully expressed in the yeast strains. For some of the receptors we observed a halo structure with FLAG-tag localization, this points to localization of the receptor into the membrane. <br><br />
When inducing the niacin receptor strain with niacin we observed that the flow cytometry peak of the induced receptor strain was shifted to a lower fluorescent intensity. This might be an indication that the cells stopped growing and thus there was a reaction on the ligand! For the banana receptor strains we had difficulties with the DNA staining in combination with the ligand Isoamyl acetate. <br />
<br />
<br/><br/><br />
<br />
<a name="P5"><h2>Recommendations</h2> </a><br />
<p>During growth of yeast cells transformed with the expression vector we observed two things: not all the cells maintain the right plasmid and the cells grew slower than wild type cells. A reason for this could be that the expression of the receptor is disadvantageously for the cells. Therefor we recommend for future work to use an inducible promoter instead of a strong constitutive promoter. In that case one can make the yeast cells expressing the receptor just before testing the strain.</p><br/><br />
<a name="P9"><h2>References</h2> </a><br />
<h6><br />
[1] Haiqing Zhao, Lidija Ivic, Joji M. Otaki, Mitsuhiro Hashimoto, Katsuhiro Mikoshiba, Stuart Firestein*Functional Expression of a Mammalian Odorant Receptor, Science 279, 237 (1998)<br/><br />
[2] Venkat Radhika, Tassula Proikas-Cezanne, Muralidharan Jayaraman, Djamila Onesime, Ji Hee Ha &Danny N Dhanasekaran, Chemical sensing of DNT by engineered olfactory yeast strain Nature Chemical Biology 3 (2007) <br/><br />
[3] Jasmina Minic, Marie-annick Persuy, Elodie Godel, Josiane Aioun, Ian Connerton, Roland Salesse, Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening, FEBS Journal (2005)<br/><br />
[4] Brown et al, Functional coupling of mammalian receptors to the yeast mating pathway using novel yeast/mammalian G protein a-subunit chimeras, Yeast (2000)<br/><br />
[5] Matthias Versele, Katleen Lemaire, and Johan M. Thevelein, Sex and sugar in yeast: two distinct GPCR systems, EMBO Rep. 2001<br/><br />
[6] Dietmar Krautwurst, King-Wai Yau, and Randall R. Reed, Identification of Ligands for Olfactory Receptors by Functional Expression of a Receptor Library, Cell (1998)<br/><br />
[7] Venkat Radhika, Tassula Proikas-Cezanne, Muralidharan Jayaraman, Djamila Onesime, Ji Hee Ha & Danny N Dhanasekaran, Chemical sensing of DNT by engineered olfactory yeast strain, Nature Chemical biology (2007)<br/><br />
[8] Alan Wise, Steven M. Foord, Neil J. Fraser, Ashley A. Barnes,e Nabil Elshourbagy, Michelle Eilert,g Diane M. Ignarg Paul R. Murdock, Klaudia Steplewski,h Andrew Green,Andrew J. Brown, Simon J. Dowell, Philip G. Szekeres, David G. Hassall, Fiona H. Marshall,a, j Shelagh Wilson, and Nicholas B. Pike<br />
Molecular Identification of High and Low Affinity Receptors for Nicotinic Acid, The journal of biological chemistry (2003) <br/><br />
[9] Georgies F. Mgode Bart J. Weetjens Thorben Nawrath, Christophe Cox, Maureen Jubitana, Robert S. Machang’, Stephan Cohen-Bacrie,e Marielle Bedotto, Michel Drancourt,e Stefan Schulz and Stefan H. E. Kaufmann, Diagnosis of Tuberculosis by Trained African Giant Pouched Rats and Confounding Impact of Pathogens and Microflora of the Respiratory Tract, Journal of clinical microbiology (2011) <br/><br />
[10] Mona Syhre, Stephen T. Chambers, The scent of Mycobacterium tuberculosis, Elsevier (2008)<br/><br />
[11 Valery Matarazzo, Olivier Clot-Faybesse, Brice Marcet, Gaelle Guiraudie-Capraz, Boriana Atanasova, Gerard Devauchelle, Martine Cerutti, Patrick Etievant and Catherine Ronin,Functional Characterization of Two Human Olfactory Receptors Expressed in the Baculovirus Sf9 Insect Cell System, Chem. Senses (2005).</h6><br/><br />
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<h2>Content</h2> <br />
<a href="#P10"> Introduction</a><br><br />
<a href="#P9">Parts </a><br><br />
<a href="#P8">Results </a><br><br />
<a href="#P7"> Conclusions</a><br><br />
<a href="#P6"> Recommendations </a><br><br />
<a href="#P5">References</a><br><br />
<br />
<a name="P10"> <br><h2>Introduction</h2> </a><br />
<p>There are two sexes of yeast cells, MATa and MATα. Whenever pheromones of the opposite sex are bound to the specific G-protein coupled receptors the MAP kinase cascade is turned on, leading to induction of mating genes such as <i>FUS1</i>. If a reporter gene as EGFP is coupled to the <i>FUS1</i> promoter the <i>FUS1</i> MAP kinase cascade can be characterized. <br />
The main questions are: What is the sensitivity of the <i>FUS1-EGFP</i> reporter, does the <i>FUS1-EGFP</i> reporter give a quantitative response, hoe long does it take before you can see a response? To answer this questions, <i>YEGFP</i> (Yeast Enhanced GFP) is cloned behind the <i>FUS1</i> promoter and the native yeast receptors are induced by alpha pheromones. With fluorometry measurement techniques we are able to see qualitative and quantitative response in time. </p><br />
<img src="https://static.igem.org/mediawiki/igem.org/a/a2/Reportersuperchill.png" width="40%" height="35%"<br />
<h6>Schematic overview of the <i>FUS1-EGFP</i> reporter</h6><br />
<a name="P9"> <h2>Parts</h2> </a><br />
<p>The <i>FUS1pr-EGFP</i> construct was designed and ordered at a synthesizing company. However the company synthesized the construct with a single nucleotide deletion in EGFP gene and therefore we cloned another EGFP behind the FUS1 promoter. The EGFP that is used is obtained from the <a href="http://www.addgene.org/14196/">pAG416GPD-ccdB-EGFP</a> plasmid (kindly provided by Harmen van Rossum from Delft University of Technology). The plasmid construct was obtained by restriction and ligation in the pRSII415 expression vector. The following biobrick was created:<br/> <br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775004 " target="_blank">BBa_K775004</a><br/></p><br/> <br />
<td> <img src="https://static.igem.org/mediawiki/igem.org/d/dd/Reporter.png" align="middle" width="500"><br/> <br />
<a name="P8"> <h2>Results</h2><a name="A4"> </a><br />
<h3>Fluorometer experiment</h3><a name="A2"> </a><br />
<h6>Setup</h6><br />
<p>Exponential growth phase cells were put into a 96 well plate and cells are induced with alpha pheromone. Cells are mixed and OD600 signal and GFP signal (excitation 485/20, emission 590/35) is measured every 1.40 minutes.</p><br />
<h6>Outcome</h6><br />
<img src="https://static.igem.org/mediawiki/igem.org/f/f4/FUS1fluorometer.png" border="0" width="80%" height="45%"><br><br />
<h6>Fluorometer data of <i>S. cerevisiae</i> transformed with <i>FUS1pr-EGFP</i></h6><br />
<p>A GFP response is expected in yeasts transformed with <i>FUS1pr-EGFP</i> when induced with alpha pheromone. The growth curve, EGFP intensity curves and EGFP intensity divided by the growth are shown in figure. Here can be observed that during time interval t=0 until t=3 Intensity relative to growth significantly increased for concentrations of 2 μM and 20 μM. For the 200 nM the peak level is at t=2. After this, the intensity decreases to normal again. Interesting is that there can be found an almost linear correlation between GFP intensity and growth for lower concentrations (seen as lines in the lower graph).</p><br />
<h3>Flow cytometry experiment</h3><br />
<h6>Setup</h6><br />
<p>When cells were in exponential phase they were induced with alpha pheromone. Cells where then measured with a Cytek FACScan. Graphs were analyzed with Flowjo.<br />
<h6>Outcome</h6><br />
<img src="https://static.igem.org/mediawiki/igem.org/0/07/FUSGFPFACS.jpg" border="0" width="90%" height="60%"><br><h6>The upper part shows Side Scattering versus fluorescence intensity. Side Scattering is an indication of the morphological structure of the cells. The lower part shows a histogram of the fluorescence intensity distribution.</h6><br />
In the figure a signal intensity shift can be observed from the I=3*10^2 towards I=2*10^3 between the WT strain induced with alpha pheromone and the <i>FUS1pr-EGFP</i> strain induced with alpha pheromone (2nd and 3rd column). This indicates that yeast cells react to the alpha pheromone with a fluorescent signal. If we look at the non induced <i>FUS1pr-EGFP</i> strain (first column) we can see that a small region correlates with higher intensity (thus EGFP expression). This is probably the signal noise of the <i>FUSpr1-EGFP</i> (leakiness). </p><br/><br />
<br><br />
<h3>Variability of the receptor: single cell microscopy</h3><br />
<h6>Description</h6><br />
With single cell microscopy we monitor the behaviour of the cellular response towards a ligand in time. For this complex process of monitoring a separate page is introduced: <a href="https://2012.igem.org/Team:TU-Delft/informationtheory"> Information processing module </a>.<br><br />
An example is shown of the process imaging which is done:<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/9/90/BF_cell_indexing.png" width="500" align="center"/><br><h6> image of Saccharomyces cerevisae, masked with tracking numbers which follow the cells over time</h6><br><br />
<br />
<a name="P7"> <h2>Conclusions</h2> </a><br />
<p> An increase in EGFP expression can be seen when yeast cells are induced with concentrations of 200 nM 2 μM and 20 μM alpha pheromone. The peak intensity of alpha pheromone addition occurs on t=2-3 hours after induction. Further promoter leakiness can be estimated to be 4%, but this is really dependent of the type of vector used and whether plasmid or chromosomal integration is chosen.</p><br/><br />
<a name="P16"> <h2>Recommendations</h2> </a><br />
<p>Further investigation should be done on fine-tuning the promoter affinity by varying the sequence of the <i>STE12p</i> docking sites and experimental conditions by estimating influencing parameters during an experiment. This includes: estimating alpha pheromone degradation rate/cell.</p><br/><br />
<br/><br />
<br />
<a name="P5"> <h2>references</h2> </a><br />
<h6>[1] Matthias Versele, Katleen Lemaire, and Johan M. Thevelein, Sex and sugar in yeast: two distinct GPCR systems, EMBO Rep. (2001<br/><br />
[2]Jasmina Minic, Marie-annick Persuy, Elodie Godel, Josiane Aioun, Ian Connerton, Roland Salesse, Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening, FEBS Journal (2005)<br/><br />
[3] Matthias Versele, Katleen Lemaire, and Johan M. Thevelein, Sex and sugar in yeast: two distinct GPCR systems, EMBO Rep. (2001)</h6><br />
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<a href="#P7"> Conclusions</a><br><br />
<a href="#P6"> Recommendations </a><br><br />
<a href="#P5">References</a><br><br />
<br />
<a name="P10"> <br><h2>Introduction</h2> </a><br />
<p>There are two sexes of yeast cells, MATa and MATα. Whenever pheromones of the opposite sex are bound to the specific G-protein coupled receptors the MAP kinase cascade is turned on, leading to induction of mating genes such as <i>FUS1</i>. If a reporter gene as EGFP is coupled to the <i>FUS1</i> promoter the <i>FUS1</i> MAP kinase cascade can be characterized. <br />
The main questions are: What is the sensitivity of the <i>FUS1-EGFP</i> reporter, does the <i>FUS1-EGFP</i> reporter give a quantitative response, hoe long does it take before you can see a response? To answer this questions, <i>YEGFP</i> (Yeast Enhanced GFP) is cloned behind the <i>FUS1</i> promoter and the native yeast receptors are induced by alpha pheromones. With fluorometry measurement techniques we are able to see qualitative and quantitative response in time. </p><br />
<img src="https://static.igem.org/mediawiki/igem.org/a/a2/Reportersuperchill.png" width="40%" height="35%"<br />
<h6>Schematic overview of the <i>FUS1-EGFP</i> reporter</h6><br />
<a name="P9"> <h2>Parts</h2> </a><br />
<p>The <i>FUS1pr-EGFP</i> construct was designed and ordered at a synthesizing company. However the company synthesized the construct with a single nucleotide deletion in EGFP gene and therefore we cloned another EGFP behind the FUS1 promoter. The EGFP that is used is obtained from the <a href="http://www.addgene.org/14196/">pAG416GPD-ccdB-EGFP</a> plasmid (kindly provided by Harmen van Rossum from Delft University of Technology). The plasmid construct was obtained by restriction and ligation in the pRSII415 expression vector. The following biobrick was created:<br/> <br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775004 " target="_blank">BBa_K775004</a><br/></p><br/> <br />
<td> <img src="https://static.igem.org/mediawiki/igem.org/d/dd/Reporter.png" align="middle" width="500"><br/> <br />
<a name="P8"> <h2>Results</h2><a name="A4"> </a><br />
<h3>Fluorometer experiment</h3><a name="A2"> </a><br />
<h6>Setup</h6><br />
<p>Exponential growth phase cells were put into a 96 well plate and cells are induced with alpha pheromone. Cells are mixed and OD600 signal and GFP signal (excitation 485/20, emission 590/35) is measured every 1.40 minutes.</p><br />
<h6>Outcome</h6><br />
<img src="https://static.igem.org/mediawiki/igem.org/f/f4/FUS1fluorometer.png" border="0" width="80%" height="45%"><br><br />
<h6>Fluorometer data of <i>S. cerevisiae</i> transformed with <i>FUS1pr-EGFP</i></h6><br />
<p>A GFP response is expected in yeasts transformed with <i>FUS1pr-EGFP</i> when induced with alpha pheromone. The growth curve, EGFP intensity curves and EGFP intensity divided by the growth are shown in figure. Here can be observed that during time interval t=0 until t=3 Intensity relative to growth significantly increased for concentrations of 2 μM and 20 μM. For the 200 nM the peak level is at t=2. After this, the intensity decreases to normal again. Interesting is that there can be found an almost linear correlation between GFP intensity and growth for lower concentrations (seen as lines in the lower graph).</p><br />
<h3>Flow cytometry experiment</h3><br />
<h6>Setup</h6><br />
<p>When cells were in exponential phase they were induced with alpha pheromone. Cells where then measured with a Cytek FACScan. Graphs were analyzed with Flowjo.<br />
<h6>Outcome</h6><br />
<img src="https://static.igem.org/mediawiki/igem.org/0/07/FUSGFPFACS.jpg" border="0" width="90%" height="60%"><br><h6>The upper part shows Side Scattering versus fluorescence intensity. Side Scattering is an indication of the morphological structure of the cells. The lower part shows a histogram of the fluorescence intensity distribution.</h6><br />
In the figure a signal intensity shift can be observed from the I=3*10^2 towards I=2*10^3 between the WT strain induced with alpha pheromone and the <i>FUS1pr-EGFP</i> strain induced with alpha pheromone (2nd and 3rd column). This indicates that yeast cells react to the alpha pheromone with a fluorescent signal. If we look at the non induced <i>FUS1pr-EGFP</i> strain (first column) we can see that a small region correlates with higher intensity (thus EGFP expression). This is probably the signal noise of the <i>FUSpr1-EGFP</i> (leakiness). </p><br/><br />
<br><br />
<h3>Variability of the receptor: single cell microscopy</h3><br />
<h6>Description</h6><br />
With single cell microscopy we monitor the behaviour of the cellular response towards a ligand in time. For this complex process of monitoring a separate page is introduced: <a href="https://2012.igem.org/Team:TU-Delft/informationtheory"> Information processing module </a>.<br><br />
An example is shown of the process imaging which is done:<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/f/f1/BF_Position2_time001.tif.out.jpg" width="500" align="center"/><br><h6> image of Saccharomyces cerevisae, masked with tracking numbers which follow the cells over time</h6><br><br />
<br />
<a name="P7"> <h2>Conclusions</h2> </a><br />
<p> An increase in EGFP expression can be seen when yeast cells are induced with concentrations of 200 nM 2 μM and 20 μM alpha pheromone. The peak intensity of alpha pheromone addition occurs on t=2-3 hours after induction. Further promoter leakiness can be estimated to be 4%, but this is really dependent of the type of vector used and whether plasmid or chromosomal integration is chosen.</p><br/><br />
<a name="P16"> <h2>Recommendations</h2> </a><br />
<p>Further investigation should be done on fine-tuning the promoter affinity by varying the sequence of the <i>STE12p</i> docking sites and experimental conditions by estimating influencing parameters during an experiment. This includes: estimating alpha pheromone degradation rate/cell.</p><br/><br />
<br/><br />
<br />
<a name="P5"> <h2>references</h2> </a><br />
<h6>[1] Matthias Versele, Katleen Lemaire, and Johan M. Thevelein, Sex and sugar in yeast: two distinct GPCR systems, EMBO Rep. (2001<br/><br />
[2]Jasmina Minic, Marie-annick Persuy, Elodie Godel, Josiane Aioun, Ian Connerton, Roland Salesse, Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening, FEBS Journal (2005)<br/><br />
[3] Matthias Versele, Katleen Lemaire, and Johan M. Thevelein, Sex and sugar in yeast: two distinct GPCR systems, EMBO Rep. (2001)</h6><br />
<br />
</div><br />
<img src="https://static.igem.org/mediawiki/igem.org/3/37/Footer_2.jpg" align="middle" width="690"><br />
<a href='https://2012.igem.org/Main_Page' target="_blank"><div id='logo_igem2'></div><a/></body></html></div>MarkWeijershttp://2012.igem.org/Team:TU-Delft/part2Team:TU-Delft/part22012-10-27T03:43:06Z<p>MarkWeijers: </p>
<hr />
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{{:Team:TUDelft/Scripts}}{{:Team:TUDelft/CSS}}{{:Team:TUDelft/menu}}<br />
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<img src="https://static.igem.org/mediawiki/igem.org/f/fd/Reporternieuw.jpg" align="middle" width="100%"><br />
<br />
<br />
<h2>Content</h2> <br />
<a href="#P10"> Introduction</a><br><br />
<a href="#P9">Parts </a><br><br />
<a href="#P8">Results </a><br><br />
<a href="#P7"> Conclusions</a><br><br />
<a href="#P6"> Recommendations </a><br><br />
<a href="#P5">References</a><br><br />
<br />
<a name="P10"> <br><h2>Introduction</h2> </a><br />
<p>There are two sexes of yeast cells, MATa and MATα. Whenever pheromones of the opposite sex are bound to the specific G-protein coupled receptors the MAP kinase cascade is turned on, leading to induction of mating genes such as <i>FUS1</i>. If a reporter gene as EGFP is coupled to the <i>FUS1</i> promoter the <i>FUS1</i> MAP kinase cascade can be characterized. <br />
The main questions are: What is the sensitivity of the <i>FUS1-EGFP</i> reporter, does the <i>FUS1-EGFP</i> reporter give a quantitative response, hoe long does it take before you can see a response? To answer this questions, <i>YEGFP</i> (Yeast Enhanced GFP) is cloned behind the <i>FUS1</i> promoter and the native yeast receptors are induced by alpha pheromones. With fluorometry measurement techniques we are able to see qualitative and quantitative response in time. </p><br />
<img src="https://static.igem.org/mediawiki/igem.org/a/a2/Reportersuperchill.png" width="40%" height="35%"<br />
<h6>Schematic overview of the <i>FUS1-EGFP</i> reporter</h6><br />
<a name="P9"> <h2>Parts</h2> </a><br />
<p>The <i>FUS1pr-EGFP</i> construct was designed and ordered at a synthesizing company. However the company synthesized the construct with a single nucleotide deletion in EGFP gene and therefore we cloned another EGFP behind the FUS1 promoter. The EGFP that is used is obtained from the <a href="http://www.addgene.org/14196/">pAG416GPD-ccdB-EGFP</a> plasmid (kindly provided by Harmen van Rossum from Delft University of Technology). The plasmid construct was obtained by restriction and ligation in the pRSII415 expression vector. The following biobrick was created:<br/> <br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775004 " target="_blank">BBa_K775004</a><br/></p><br/> <br />
<td> <img src="https://static.igem.org/mediawiki/igem.org/d/dd/Reporter.png" align="middle" width="500"><br/> <br />
<a name="P8"> <h2>Results</h2><a name="A4"> </a><br />
<h3>Fluorometer experiment</h3><a name="A2"> </a><br />
<h6>Setup</h6><br />
<p>Exponential growth phase cells were put into a 96 well plate and cells are induced with alpha pheromone. Cells are mixed and OD600 signal and GFP signal (excitation 485/20, emission 590/35) is measured every 1.40 minutes.</p><br />
<h6>Outcome</h6><br />
<img src="https://static.igem.org/mediawiki/igem.org/f/f4/FUS1fluorometer.png" border="0" width="80%" height="45%"><br><br />
<h6>Fluorometer data of <i>S. cerevisiae</i> transformed with <i>FUS1pr-EGFP</i></h6><br />
<p>A GFP response is expected in yeasts transformed with <i>FUS1pr-EGFP</i> when induced with alpha pheromone. The growth curve, EGFP intensity curves and EGFP intensity divided by the growth are shown in figure. Here can be observed that during time interval t=0 until t=3 Intensity relative to growth significantly increased for concentrations of 2 μM and 20 μM. For the 200 nM the peak level is at t=2. After this, the intensity decreases to normal again. Interesting is that there can be found an almost linear correlation between GFP intensity and growth for lower concentrations (seen as lines in the lower graph).</p><br />
<h3>Flow cytometry experiment</h3><br />
<h6>Setup</h6><br />
<p>When cells were in exponential phase they were induced with alpha pheromone. Cells where then measured with a Cytek FACScan. Graphs were analyzed with Flowjo.<br />
<h6>Outcome</h6><br />
<img src="https://static.igem.org/mediawiki/igem.org/0/07/FUSGFPFACS.jpg" border="0" width="90%" height="60%"><br><h6>The upper part shows Side Scattering versus fluorescence intensity. Side Scattering is an indication of the morphological structure of the cells. The lower part shows a histogram of the fluorescence intensity distribution.</h6><br />
In the figure a signal intensity shift can be observed from the I=3*10^2 towards I=2*10^3 between the WT strain induced with alpha pheromone and the <i>FUS1pr-EGFP</i> strain induced with alpha pheromone (2nd and 3rd column). This indicates that yeast cells react to the alpha pheromone with a fluorescent signal. If we look at the non induced <i>FUS1pr-EGFP</i> strain (first column) we can see that a small region correlates with higher intensity (thus EGFP expression). This is probably the signal noise of the <i>FUSpr1-EGFP</i> (leakiness). </p><br/><br />
<br><br />
<h3>Variability of the receptor: single cell microscopy</h3><br />
<h6>Description</h6><br />
With single cell microscopy we monitor the behaviour of the cellular response towards a ligand in time. For this complex process of monitoring a separate page is introduced: <a href="https://2012.igem.org/Team:TU-Delft/informationtheory"> Information processing module </a>.<br><br />
An example is shown of the process imaging which is done:<br><br />
<img src="https://static.igem.org/mediawiki/2012/f/f1/BF_Position2_time001.tif.out.jpg" width="500" align="center"/><br><br><br />
<br />
<a name="P7"> <h2>Conclusions</h2> </a><br />
<p> An increase in EGFP expression can be seen when yeast cells are induced with concentrations of 200 nM 2 μM and 20 μM alpha pheromone. The peak intensity of alpha pheromone addition occurs on t=2-3 hours after induction. Further promoter leakiness can be estimated to be 4%, but this is really dependent of the type of vector used and whether plasmid or chromosomal integration is chosen.</p><br/><br />
<a name="P16"> <h2>Recommendations</h2> </a><br />
<p>Further investigation should be done on fine-tuning the promoter affinity by varying the sequence of the <i>STE12p</i> docking sites and experimental conditions by estimating influencing parameters during an experiment. This includes: estimating alpha pheromone degradation rate/cell.</p><br/><br />
<br/><br />
<br />
<a name="P5"> <h2>references</h2> </a><br />
<h6>[1] Matthias Versele, Katleen Lemaire, and Johan M. Thevelein, Sex and sugar in yeast: two distinct GPCR systems, EMBO Rep. (2001<br/><br />
[2]Jasmina Minic, Marie-annick Persuy, Elodie Godel, Josiane Aioun, Ian Connerton, Roland Salesse, Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening, FEBS Journal (2005)<br/><br />
[3] Matthias Versele, Katleen Lemaire, and Johan M. Thevelein, Sex and sugar in yeast: two distinct GPCR systems, EMBO Rep. (2001)</h6><br />
<br />
</div><br />
<img src="https://static.igem.org/mediawiki/igem.org/3/37/Footer_2.jpg" align="middle" width="690"><br />
<a href='https://2012.igem.org/Main_Page' target="_blank"><div id='logo_igem2'></div><a/></body></html></div>MarkWeijershttp://2012.igem.org/Team:TU-Delft/part2Team:TU-Delft/part22012-10-27T03:41:52Z<p>MarkWeijers: </p>
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<br />
<br />
<h2>Content</h2> <br />
<a href="#P10"> Introduction</a><br><br />
<a href="#P9">Parts </a><br><br />
<a href="#P8">Results </a><br><br />
<a href="#P7"> Conclusions</a><br><br />
<a href="#P6"> Recommendations </a><br><br />
<a href="#P5">References</a><br><br />
<br />
<a name="P10"> <br><h2>Introduction</h2> </a><br />
<p>There are two sexes of yeast cells, MATa and MATα. Whenever pheromones of the opposite sex are bound to the specific G-protein coupled receptors the MAP kinase cascade is turned on, leading to induction of mating genes such as <i>FUS1</i>. If a reporter gene as EGFP is coupled to the <i>FUS1</i> promoter the <i>FUS1</i> MAP kinase cascade can be characterized. <br />
The main questions are: What is the sensitivity of the <i>FUS1-EGFP</i> reporter, does the <i>FUS1-EGFP</i> reporter give a quantitative response, hoe long does it take before you can see a response? To answer this questions, <i>YEGFP</i> (Yeast Enhanced GFP) is cloned behind the <i>FUS1</i> promoter and the native yeast receptors are induced by alpha pheromones. With fluorometry measurement techniques we are able to see qualitative and quantitative response in time. </p><br />
<img src="https://static.igem.org/mediawiki/igem.org/a/a2/Reportersuperchill.png" width="40%" height="35%"<br />
<h6>Schematic overview of the <i>FUS1-EGFP</i> reporter</h6><br />
<a name="P9"> <h2>Parts</h2> </a><br />
<p>The <i>FUS1pr-EGFP</i> construct was designed and ordered at a synthesizing company. However the company synthesized the construct with a single nucleotide deletion in EGFP gene and therefore we cloned another EGFP behind the FUS1 promoter. The EGFP that is used is obtained from the <a href="http://www.addgene.org/14196/">pAG416GPD-ccdB-EGFP</a> plasmid (kindly provided by Harmen van Rossum from Delft University of Technology). The plasmid construct was obtained by restriction and ligation in the pRSII415 expression vector. The following biobrick was created:<br/> <br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775004 " target="_blank">BBa_K775004</a><br/></p><br/> <br />
<td> <img src="https://static.igem.org/mediawiki/igem.org/d/dd/Reporter.png" align="middle" width="500"><br/> <br />
<a name="P8"> <h2>Results</h2><a name="A4"> </a><br />
<h3>Fluorometer experiment</h3><a name="A2"> </a><br />
<h6>Setup</h6><br />
<p>Exponential growth phase cells were put into a 96 well plate and cells are induced with alpha pheromone. Cells are mixed and OD600 signal and GFP signal (excitation 485/20, emission 590/35) is measured every 1.40 minutes.</p><br />
<h6>Outcome</h6><br />
<img src="https://static.igem.org/mediawiki/igem.org/f/f4/FUS1fluorometer.png" border="0" width="80%" height="45%"><br><br />
<h6>Fluorometer data of <i>S. cerevisiae</i> transformed with <i>FUS1pr-EGFP</i></h6><br />
<p>A GFP response is expected in yeasts transformed with <i>FUS1pr-EGFP</i> when induced with alpha pheromone. The growth curve, EGFP intensity curves and EGFP intensity divided by the growth are shown in figure. Here can be observed that during time interval t=0 until t=3 Intensity relative to growth significantly increased for concentrations of 2 μM and 20 μM. For the 200 nM the peak level is at t=2. After this, the intensity decreases to normal again. Interesting is that there can be found an almost linear correlation between GFP intensity and growth for lower concentrations (seen as lines in the lower graph).</p><br />
<h3>Flow cytometry experiment</h3><br />
<h6>Setup</h6><br />
<p>When cells were in exponential phase they were induced with alpha pheromone. Cells where then measured with a Cytek FACScan. Graphs were analyzed with Flowjo.<br />
<h6>Outcome</h6><br />
<img src="https://static.igem.org/mediawiki/igem.org/0/07/FUSGFPFACS.jpg" border="0" width="90%" height="60%"><br><h6>The upper part shows Side Scattering versus fluorescence intensity. Side Scattering is an indication of the morphological structure of the cells. The lower part shows a histogram of the fluorescence intensity distribution.</h6><br />
In the figure a signal intensity shift can be observed from the I=3*10^2 towards I=2*10^3 between the WT strain induced with alpha pheromone and the <i>FUS1pr-EGFP</i> strain induced with alpha pheromone (2nd and 3rd column). This indicates that yeast cells react to the alpha pheromone with a fluorescent signal. If we look at the non induced <i>FUS1pr-EGFP</i> strain (first column) we can see that a small region correlates with higher intensity (thus EGFP expression). This is probably the signal noise of the <i>FUSpr1-EGFP</i> (leakiness). </p><br/><br />
<a name="P7"> <h2>Conclusions</h2> </a><br />
<p> An increase in EGFP expression can be seen when yeast cells are induced with concentrations of 200 nM 2 μM and 20 μM alpha pheromone. The peak intensity of alpha pheromone addition occurs on t=2-3 hours after induction. Further promoter leakiness can be estimated to be 4%, but this is really dependent of the type of vector used and whether plasmid or chromosomal integration is chosen.</p><br/><br />
<a name="P16"> <h2>Recommendations</h2> </a><br />
<p>Further investigation should be done on fine-tuning the promoter affinity by varying the sequence of the <i>STE12p</i> docking sites and experimental conditions by estimating influencing parameters during an experiment. This includes: estimating alpha pheromone degradation rate/cell.</p><br/><br />
<br/><br />
<h3>Variability of the receptor: single cell microscopy</h3><br />
<h6>Description</h6><br />
With single cell microscopy we monitor the behaviour of the cellular response towards a ligand in time. For this complex process of monitoring a separate page is introduced: <a href="https://2012.igem.org/Team:TU-Delft/informationtheory"> Information processing module </a>.<br><br />
An example is shown of the process imaging which is done:<br><br />
<img src="https://static.igem.org/mediawiki/2012/f/f1/BF_Position2_time001.tif.out.jpg" width="600"/><br />
<br />
<br />
<br />
<br />
<a name="P5"> <h2>references</h2> </a><br />
<h6>[1] Matthias Versele, Katleen Lemaire, and Johan M. Thevelein, Sex and sugar in yeast: two distinct GPCR systems, EMBO Rep. (2001<br/><br />
[2]Jasmina Minic, Marie-annick Persuy, Elodie Godel, Josiane Aioun, Ian Connerton, Roland Salesse, Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening, FEBS Journal (2005)<br/><br />
[3] Matthias Versele, Katleen Lemaire, and Johan M. Thevelein, Sex and sugar in yeast: two distinct GPCR systems, EMBO Rep. (2001)</h6><br />
<br />
</div><br />
<img src="https://static.igem.org/mediawiki/igem.org/3/37/Footer_2.jpg" align="middle" width="690"><br />
<a href='https://2012.igem.org/Main_Page' target="_blank"><div id='logo_igem2'></div><a/></body></html></div>MarkWeijershttp://2012.igem.org/File:BF_Position2_time001.tif.out.jpgFile:BF Position2 time001.tif.out.jpg2012-10-27T03:40:44Z<p>MarkWeijers: uploaded a new version of &quot;File:BF Position2 time001.tif.out.jpg&quot;</p>
<hr />
<div></div>MarkWeijershttp://2012.igem.org/File:BF_Position2_time001.tif.out.jpgFile:BF Position2 time001.tif.out.jpg2012-10-27T03:40:30Z<p>MarkWeijers: </p>
<hr />
<div></div>MarkWeijershttp://2012.igem.org/File:BF_Position2_time001.tif.out.tifFile:BF Position2 time001.tif.out.tif2012-10-27T03:39:49Z<p>MarkWeijers: </p>
<hr />
<div></div>MarkWeijershttp://2012.igem.org/Team:TU-Delft/AcknowledgementsTeam:TU-Delft/Acknowledgements2012-10-27T03:34:23Z<p>MarkWeijers: </p>
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<div>{{:Team:TUDelft/Scripts}}{{:Team:TUDelft/CSS}}{{:Team:TUDelft/menu}}<br />
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<head><title>Acknowledgements</title><br />
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<div style="height:70px; width:100%;"></div> <br />
<img src="https://static.igem.org/mediawiki/igem.org/7/74/Background_greenlamp.jpg" class="bg_team"><br />
<div id="logo_ed"><a href="https://2012.igem.org/Team:TU-Delft" 'onfocus=this.blur()'><img src="https://static.igem.org/mediawiki/igem.org/8/88/Logoigemklein.png" border="0" width="100" height="100"></a></div><br />
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<img src="https://static.igem.org/mediawiki/igem.org/0/0e/Acknowledgements.jpg" align="middle" width="100%"><br />
<br />
<br />
<br />
<p><b>For the completion of this project have contributed many people. Thus, we would like to thank: </b></p><br />
<h3>Our instructors</h3><br />
<ul><br />
<li>Dr. Anne Meyer</li><br />
<li>Ing. Esengül Yildirim</li><br />
<li>Dr. Ir. Alessandro Abate</li><br />
<p>for their continuous mentorship and patience throughout the whole project</p> <br />
<br/><p> As well as our advisors that, with their knowledge in their specific<br />
field, had always a useful advice for us.</p><br />
<li>Dr. Emrah Nikerel </li><br />
<li>Dr. Aljoscha Wahl </li><br />
<li>MSc. Ilya Tkachev </li><br />
<li>MSc. Calin Plesa </li><br />
<li>MSc. Daniel Solis Escalante </li><br />
<p>and of course all of the team members for all the moments we shared, as well as Willem Noteborn, who started as an active member of our team. We thank him for the help on the brainstorming sessions.</p><br />
</ul><br />
<br />
<h3>Department of Biotechnology</h3><br />
<ul><br />
<li>Enzymology Department for hosting our team by providing lab space</LI><br />
<li>Prof. dr. Simon de Vries</li><br />
<p>all the people that made it possible to do this project by providing permits</p><br />
<li>Prof. dr. Han de Winde</li><br />
<li>Dr. Lesley Robertson</li><br />
<li>Ing. Marinka Almering</li><br />
<li>Dr. Laura Koekkoek-van der Weel for the use of fluorometer</li><br />
<li>MSc. Tim Vos for his advice on many many yeast related things</li><br />
<li>MSc. Niels Kuijpers for his advice at the brainstorming session as well as towards yeast</li><br />
<li>MSc. Harmen van Rossum for the EGFP plasmid</li><br />
<li>The kitchen: Epilena, Astrid and Jannie</li><br />
<li>Ing. Dirk Geerts and Ing. Rob Kerste and the BPT group for helping with the fermentations</li><br />
<li>Ing. Udo van Dongen for the use of the microscope</li><br />
</ul><br />
<br />
<h3>Bionanoscience Department</h3><br />
<ul><br />
<li>Dr. Daniel Lam for the use of Microscopes</li><br />
<li>MSc. Pauline van Nies for her advice towards fluorometer</li><br />
<li>Ing. Suzanne Hage for letting us experiment in her lab</li><br />
<li>Dr. Yaron Caspi for helping with microscopy</li><br />
<li>MSc. Michela de Martino for the use of NanoDrop</li><br />
<li>MSc. Felix Hol for the use of flow cytometry</li><br />
<li>MSc. Vlad Karas for help with flow cytometry</li><br />
<li>MSc. Fabai Wu for his advice at the brainstorming session as well as for fluorescent imaging</li><br />
<li>Dr. Yaron Caspi for his advice at the brainstorming session as well as for fluorescent imaging</li><br />
</ul><br />
<br />
<h3>Computer Science Department</h3><br />
<ul><br />
<p>the <a href='http://bioinformatics.tudelft.nl/' target="_blank">Bioinformatics Lab </a> <br />
<li>Prof.dr.ir. Marcel Reinders for the financial support as well as for his information about iGEM</li><br />
<li>Bastiaan van den Berg for his advice on Software usage for tertiary structure of Proteins</li> <br />
</ul><br />
<br />
<h3>Leiden University</h3><br />
<ul><br />
<p>for letting Leiden students aware of iGEM competition and thus we have Lizah in our team!</p><br />
<li>Dr.Dennis Claessen</li><br />
<li>Prof.Dr.Gilles van Wezel</li><br />
</ul><br />
<br />
<h3>Experts on the field</h3><br />
<ul><br />
<li>Dr. Mark Chee from Haase lab for proving the pRSII plasmids</li><br />
<li>Dr. Amie McClellan for advice on yeast</li><br />
<li>Dr. James Broach for advise and sending yeast strains.</li><br />
<li>Dr. Georgies F. Mgode for advice on TB sensoring</li><br />
<li>Dr. Bart J. Weetjens for the great idea to smell tuberculosis</li><br />
</ul><br />
<br />
<br />
<h3>Media</h3><br />
<ul> <br />
<li>NRC</li><br />
</ul><br />
<br />
<h3>Modeling</h3><br />
<ul><br />
<li>Dr. Cristian Picioreanu </li><br />
<li>Linda G. Otten</li><br />
<li>Dirk J. Opperman</li><br />
<li>Lykle Voort from the SARA institute</li><br />
</ul><br />
For helping with the information processing:<br />
<ul><br />
<li>Dr. Raymond Cheong</li><br />
<li>Dr. Andre Levchenko</li><br />
<li>Dr. Colman Lerner</li><br />
</ul><br />
<br />
<br />
<h3>For the professional Photoshoot and Filming</h3><br />
<ul><br />
<li>Frank Theys</li><br />
</ul><br />
<br />
<br />
<h3>Science Center</h3><br />
<p>for providing us rooms for our brainstorming sessions</p><br />
<br />
<br />
<br />
<h3>Marketing</h3><br />
<ul><br />
<p>for his advices towards Marketing</p><br />
<li>drs. Guillaume Karremans</li><br />
</ul><br />
<br />
<br />
<h3>Wiki</h3><br />
<ul><li>the iGEM team <a href='https://2011.igem.org/Team:KULeuven' target="_blank">KULeuven 2011</a> that according to their wiki we have built ours</li></ul> <br />
<br />
<h3>LLowlab</h3><br />
<p>for their invitation at the <a href='http://www.denachtvankunstenwetenschap.nl/en/' target="_blank">Night of Arts & Sciences</a> and at the <a href='http://www.llowlab.nl/' target="_blank">LLowlab</a> 2012 event. </p><br />
<ul><li> Jan Douwe Kroeske for giving us the opportunity to go to Llowlab</li><br />
<li> Noor Vos for a great Llowlab event</li><br />
<li> Janneke de Ruiter for the publicity around Llowlab</li><br />
<li> Bart van de Laar for the Night of Arts & Sciences</li></ul></p><br />
<br />
<h3>For our iGEM android app</h3><br />
<ul><li>Bas van Stein, MSc student in Computer Science at Leiden University</li></ul><br />
<br />
<h3>Family and Friends</h3><br />
<p>for their support all those months</p><br />
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</div></div>MarkWeijershttp://2012.igem.org/Team:TU-Delft/protocolsTeam:TU-Delft/protocols2012-10-27T03:07:40Z<p>MarkWeijers: </p>
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<h1>Protocols</h1> <br />
<br />
<a href="#P10"> Media</a><br/><br />
<a href="#P9"> Yeast Transformation</a><br/><br />
<a href="#P1"> Transforming One Shot® Mach1™ competent cells </a><br/><br />
<a href="#P2"> Mini-prep plasmid isolation for E. coli and S. cerevisae </a><br/><br />
<a href="#P3"> Freezing cell stocks </a><br/><br />
<a href="#P4"> Restriction enzyme digestion </a><br/><br />
<a href="#P5"> Ligation </a><br/><br />
<a href="#P6"> Running DNA Gel </a><br/><br />
<a href="#P7"> PCR </a><br/><br />
<a href="#P8">Gel Extraction</a><br/><br />
<a href="#P11">Immunofluorescence Staining</a><br/><br />
<a href="#P12">DNA Staining</a><br/><br />
<a href="#P13">Flow cytometry to look at DNA content</a><br/><br />
<a href="#P14">Flow cytometry to look at EGFP expression</a><br/><br />
<a href="#P15">Fluorimeter experiment</a><br/><br />
<a href="#P17">Growth curve experiment</a><br/><br />
<a href="#P18">Smell activity tests with petri dishes</a><br/><br />
<a href="#P16">Tips&Tricks for working with yeast</a><br/><br/><br />
<br />
<a name="P10"><h1>Media</h1></a><br />
<br/><br />
<i>DO (agar)</i><br />
<br/>For BY4741 yeast strain add to mineral media:<br/>HIS: 125 mg/liter<br/>LEU 500 mg/liter<br/>MET 100 mg/liter<br/>URA 150 mg/liter<br/>except for one which you want to select on with you auxotrophic marker.<br/><br/>Also add:<br/>2% Sucrose (w/v)<br/>2% Agarose<br/><br/>and autoclave<br/><br/><br />
<i>YPD (agar)</i><br />
<br/>http://openwetware.org/wiki/YPD <br/><br/><br />
<i>LB (agar) </i><br/><br />
<br/>http://openwetware.org/wiki/LB <br/><br/><br />
<br />
<a name="P9"><h1>Yeast Transformation</h1></a><br />
<br/><br />
<i>Materials</i><br/><br />
<ul><li>Yeast culture</li><br />
<li>single stranded carrier DNA (200 mg Salmon sperm DNA inTE)</li><br />
<li>Lithium Acetate (1.0 M)</li><br />
<li>Polyethylene Glycol 3350 (50% w/v)</li><br />
<li>DO media plates </li><br />
<li>Dropout (DO) media, lacking an amino acid of which an auxotrophic marker is added</li><br />
<li>Water bath 42 °C</li><br />
<li>Water bath 30 °C</li><br/><br/><br />
</ul><br />
<br />
<i>Protocol</i><br/><br />
<br />
<br />
<ol><li> Grow yeast culture overnight in 10 mL YPD on 30 °C</li><br />
<li> Put 0.5 mL – 1 mL into new flask with 20 mL YPD (for 2-4 transformations) and grow 4-5 hours until an OD of 0.8 is measured. The cells are now in exponential phase.</li><br />
<li> Spin the cells for 5 minutes on 3000 G and discard supernatant</li><br />
<li> Wash cells with water and spin down for 5 minutes on 3000 G</li><br />
<li> During centrifuging, make a transformation mix (TM): Per reaction add <br/><br />
<ul><li>240 µL 50% PEG</li><br />
<li>36 µL 1M Lithium Acetate solution</li><br />
<li>25 µL ssDNA, boiled for 5 minutes and then cooled on ice</li><br />
<li>49 µL water</li><br />
</ul><br/><br />
</li><br />
<li> Discard supernatant and resuspend in 0.2 mL 0.1 M Lithium Acetate solution</li><br />
<li> Do a quick centrifuge (10 s 13.000 rpm on a table top centrifuge), discard supernatant and add 80 µL 0.1 M Lithium Acetate solution. Vortex and do <u>not</u> pipette up and down</li><br />
<li>You should have ~100 µL of which 50 µL should be put in a fresh tube. This is you negative control</li><br />
<li>spin cells down again and add the mastermix</li><br />
<li>Add 1 µL target (100 ng – 1000 ng) DNA to the positive tube</li><br />
<li>Put in a 30 °C water bath for 30 minutes</li><br />
<li>Put it in a 42 °C water bath for 30 minutes</li><br />
<li>Do a quick centrifuge (10 s 13.000 rpm on a table top), remove supernatant. Add 0.2 mL water, pipet up and down and plate 150 µL and 50 µL on DO selective plates </li><br />
</ol><br/><br/><br />
<br />
<br />
<a name="P1"> <h1>Transforming One Shot® Mach1™ competent cells</h1></a><br/><br />
<br />
<br/><br />
<i>Materials:</i><br />
<br/><br />
<ul><br />
<li>- Competent cells</li><br />
<li> - SOC medium (warmed to room temperature)</li><br />
<li> - Plasmid DNA or DNA ligation mix</li><br />
<li> - LB agar plates containing 15-100 ?g/mL antibiotic of choice, pre-warmed to 37 °C </li><br />
<li> - water bath at 42 °C </li><br />
<li> - shaking incubator at 37 °C. </li><br />
</ul><br />
<br/><br />
<i>Protocol:</i><br />
<br/><br />
<ol><li>Add 50-100 ng DNA into a 20 ?L competent E.coli, and mix gently. Do not mix by pipetting up and down!</li><br />
<li>Incubate tube vial on ice for 30 minutes</li><br />
<li>Heat-shocks the cells for 30 seconds at 42 °C without shaking</li><br />
<li>Immediately transfer the tubes back to ice for 2 minutes</li><br />
<li>Add 250 ?L of room temperature LB medium</li><br />
<li>Cap tube tightly and shake tube horizontally (225 rpm) at 37 °C for 1 hour</li><br />
<li>Plate from each tube 100 ?L on an agar plate containing antibiotic. Spin tube, discard supernatant to leave no more than 100 ?L, vortex and plate on an agar plate</li><br />
<li>Incubate plates overnight at 37 °C</li><br />
</ol><br />
<br/><br />
<br />
<br />
<br />
<a name="P2"> <h1> Qiagen Mini-prep plasmid isolation for E. coli and S. cerevisae</h1></a><br />
<br/><br />
This protocol is based on QIAGEN® Plasmid Purification Handbook.<br/><br />
<i>Materials:</i><br/><br />
<ul><br />
<li>- bacterial or yeast culture</li><br />
<li>- Qiagen colums</li><br />
<li>- buffer P1 (100 mg/mL RNAse A, 50 mM Tris/HCl, 10 mM EDTA, pH 8.0) </li><br />
<li>- buffer P2 (200 mM NaOH, 1% SDS) </li><br />
<li>- buffer P3 (3 M KAc, pH 5.5) </li><br />
<li>- buffer PE </li><br />
<li>- milliQ pH 8.0</li><br />
<li>- centrifuge</li><br />
<li>- nanodrop</li><br />
<li>- for yeast plasmid isolation: zymolyase 5 U/µl</li></ul><br/><br />
<i> Protocol:</i><br/><br />
<ol><br />
<li>Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 2–5 mL LB medium containing the appropriate selective antibiotic or selective medium. Incubate for approximately 8 h at 37°C (bacteria) or 12 h at 30°C with vigorous shaking (approx. 300 rpm)</li><br />
<li>Harvest the 5 mL bacterial cells by centrifugation at 13,000 rpm for 1 min at 20°C (microcentrifuge tube). If you wish to stop the protocol and continue later, freeze the cell pellets at –20°C</li><br />
<li>For bacteria: Resuspend pelleted bacterial cells in 250 µL Buffer P1. Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet. <br/> For yeast: Resuspend cells in 250 µL Buffer P1 with 3 µL zymolyase and incubate 1 h at 37 °C</li><br />
<li>Add 250 µL Buffer P2 and mix thoroughly by inverting the tube 4–6 times. Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. 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><br />
<li>Add 350 µL Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer N3. Large culture volumes (e.g. =5 mL) may require inverting up to 10 times. The solution should become cloudy. </li><br />
<li>Incubate at -20 °C for 15 minutes. </li><br />
<li>Centrifuge for 10 min at 13,000 rpm in a table-top microcentrifuge. A compact white pellet will form. </li><br />
<li>Apply the supernatants from step 7 to the QIAprep spin column by decanting or pipetting. </li><br />
<li>Centrifuge for 30–60 seconds. Discard the flow-through. </li><br />
<li>Wash QIAprep spin column by adding 0.75 mL Buffer PE and centrifuging for 30–60 seconds. </li><br />
<li>Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer. <br />
<b>Important:</b> Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions. </li><br />
<li>Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 30 µL Buffer EB (10 mM Tris•Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 minute in 50 C stove and centrifuge for 1 minute to obtain DNA. </li><br />
<li>Measure DNA concentration on the Nanodrop</li><br />
</ol><br/><br />
<br />
<br />
<br />
<br />
<a name="P3"> <h1>Freezing cell stocks</h1></a><br />
<br/><br />
<i>Materials:</i> <br />
<ul><br />
<li>- bacterial culture </li><br />
<li>- Growth medium</li><br />
<li>- 80% glycerol</li><br />
<li>- centrifuge</li><br />
</ul><br />
<br/><br />
<br />
<i>Protocol:</i><br/><br />
<ol><br />
<li>Take 5 mL bacterial cells from the Erlenmeyer of a freshly grown culture and spin in a 15 mL tube for 10 minutes at 2.000 rpm (Eppendorf centrifuge)</li><br />
<li>Decant the supernatant without disturbing the pellet</li><br />
<li>Pipet on the pellet 0.5 ml of appropriate medium and 0.5 mL 80% glycerol and mix by vortexing and save in -80 °C freezer</li><br />
</ol><br />
<br/><br />
<br />
<br />
<br />
<br />
<a name="P4"> <h1> Restriction enzyme digestion </h1></a><br />
<br/><br />
<i>Materials:</i><br/><br />
<ul><br />
<li>- plasmid DNA or PCR product </li><br />
<li>- restriction enzymes (Roche and BioLabs) </li><br />
<li>- buffer (10x) </li><br />
<li>- H2O</li><br />
<li>water bath at 37 °C</li><br />
<li>- heat block or water bath at 65 °C</li></ul><br/><br />
<i>Protocol:</i><br/><br />
Digestions (cutting plasmid DNA) were performed at the appropriate temperature with the appropriate buffer in the appropriate concentration, according to the supplier. With double restriction, use bigger volume (~ 50 µL) and we found out that adding BSA altered performance greatly.<br/><br/><br />
<br/>Reaction for one sample:<br/><br />
DNA × µL (up to 1,0 µg)<br/><br />
Buffer (10×) × µL (1×))<br/><br />
Restriction enzymes × µL (5 units/µg DNA = 1 µL) )<br/><br />
H2O × µL<br/><br />
tot volume 20-25 µL<br />
<br/><br/><br />
Incubate for (at least) one hour at 37 °C. Inactivate the restriction endonucleases by heat, incubation at 65 °C for 10 minutes and centrifuge shortly.<br/><br />
<br/><br />
<i><u>Used Buffers:</u></i><br/><br />
Buffer H (Roche): 50 mM Tris-HCl, 1 M NaCl, 100 mM MgCl2, 10 mM DTE, pH 7.5 at 37 °C<br/><br />
Buffer M (Roche): 100 mM Tris-HCl, 500 mM NaCI, 100 mM MgCl2, 10 mM DTE, pH 7.5 at 37 °C<br/><br />
Buffer 1 (BioLabs): 10 mM Bis-Tris-Propane-HCl, 10 mM MgCl2, 1 mM DTE,pH 7.0 at 25°C<br/><br />
Buffer 2 (BioLabs): 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTE, pH 7.9 at 25°C<br/><br />
Buffer 3 (BioLabs): 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 1 mM DTE, pH 7.9 at 25°C<br/><br />
Buffer 4 (BioLabs): 50 mM CH3CO2K, 20 mM TAE, 10 mM Mg(CH3COO)2, 1 mM DTE, pH 7.9 at 25°C<br/><br/><br />
Note: some of the restriction enzymes of New England BioLabs required the addition of 100 µg/mL BSA)<br/><br />
<br />
<br><br />
<br />
<br />
<br />
<a name="P5"> <h1>Ligation</h1></a><br />
<br/><br />
<i>Materials:</i><br />
<ul><br />
<li>- digested plasmid DNA or PCR product</li><br />
<li>- T4 ligation buffer (10x) (Fermentas)</li><br />
<li>- T4 ligase (Fermentas)</li><br />
<li>- H2O</li><br />
<li>water bath at 16 °C</li><br />
</ul><br />
<br/><br />
<i>Protocol:</i><br />
Ligations (pasting plasmid DNA) were performed at the appropriate temperature with the appropriate buffer in the appropriate concentration, according to the supplier. We encountered different tactics for ligation. Usually it comes down to keeping it ~16 °C for at least 3 hours.<br />
<br/><br/><br />
<br />
Reaction for one sample:<br/><br />
DNA insert × µL<br/><br />
DNA vector × µL <br/><br />
T4 Ligation buffer (10×) × µL (for 1×)<br/><br />
T4 Ligase 1.0 µL <br/><br />
H2O × µL<br/><br />
tot. volume 10-15 µL<br/><br/><br />
<br />
The final concentration is preferably ~100 ng/µL. Smaller volumes are preferred and when DNA is at low concentration, try to evaporate water using a vacuum. Incubate at 16 °C for at least 3 hours or keep in an ice box in a floatie overnight. In the morning you find eppendorf tubes floating in water which had a temperature gradient overnight. For transformation use circa half of the ligation mix. <br/><br/><br />
<br />
<br />
<br />
<br />
<a name="P6"> <h1>Running a DNA gel</h1></a><br />
<br/><br />
<i>Materials:</i><br />
<ul><br />
<li>- Agarose </li><br />
<li>- TAE 1x </li><br />
<li>- SybrSafe DNA stain</li><br />
<li>- Loading Dye</li><br />
<li>- DNA ladder (Smartladder)</li><br />
<li>- DNA electrophoresis machine</li><br />
</ul><br />
<br/><br />
<i>Protocol:</i><br/><br />
<ol><br />
<li> Dissolve agarose (w/v 0.6% for separating long DNA pieces (>10 kbp), 1% for separating shorter pieces) in 1x TAE by microwaving</li><br />
<li> Close sides of electrophoresis tray (scotch tape works fine) and add comb</li><br />
<li> Let solution cool and add 5 µl Sybrsafe to an empty electrophoresis tray (small gels) 10-12 µl Sybrsafe for larger gels </li><br />
<li> Pour gel until a height of ~0.5 cm. Mix and remove bubbles with pipet tip (fast! It hardens quickly)</li><br />
<li> Put tray into electrophoresis casing and add TAE until a small layer above the gel can be seen. Remove comb</li><br />
<li>Add 1 µl loading dye to 5 µl sample, mix and load in the gel. Also add 5 µl smartladder for your reference</li><br />
<li>Run gel on 80 V (long run)- 110 V (short run, mostly for a ‘fast check’) for ~40-60 minutes, dependant of gel size, separation acquisition and voltage.</li><br />
</ol><br/><br />
For information about the smartladder, <a href=" https://secure.eurogentec.com/product/research-smartladder.html">Smartladder specifications</a>.<br />
<br/><br/><br />
<br />
<br />
<br />
<a name="P7"> <h1>PCR</h1></a><br />
<br/><br />
<i>Materials:</i><br/><br />
For PCR with minimal errors, pFX polymerase is used:<br />
<ul><br />
<li>Pfx polymerase (Invitrogen) </li><li><br />
10x Pfx Buffer (Invitrogen) </li><li><br />
enhancer (Invitrogen) </li><li><br />
50 mM MgSO4 (Invitrogen) </li><li><br />
10 mM dNTPs </li><br />
</ul><br />
<br/><br />
<br />
For PCR for checks of length, Taq polymerase is used, provided in Qiagen mastermix:<br/><br />
<ul><li>Mastermix </li></ul><br />
<br/><br />
Always needed:<br/><br />
<ul><br />
<li>primer solutions 5 mol/mL</li><li><br />
template DNA (plasmid at 50 pg – 1 ng/µL), or plate with colonies</li><li><br />
PCR machine</li><br />
</ul><br />
<br/><br/><br />
<br />
<br />
<i>Protocol:</i><br/><br />
First make sure that there is a PCR machine available for you. Take the solutions from the freezer and thaw them on ice.<br/><br/><br />
Preparation of reaction mixture:<br />
<ol><li>Gently vortex and briefly centrifuge all solutions after thawing</li><br />
<li>Keep solutions on ice</li><br />
<li>Add to a thin walled PCR tube, on ice the desired reaction mixture listed below.</li><br />
<li>for PCR on colonies: prick a sterile toothpick into a colony, dip it into a PCR tube and put it in 15 mL culture tube containing growth media to grow overnight for direct culturing positives.</li><br />
<li>Make sure you keep everything cool until it enters the preheated PCR machine</li></ol><br />
<br/><br />
<br />
<br />
<hr><br />
<br />
<div class=WordSection1><br />
<br />
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0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal style='line-height:normal'><st1:metricconverter<br />
ProductID="50 mM" w:st="on"><span lang=DE style='mso-ansi-language:DE'>50 <span<br />
class=SpellE>mM</span></span></st1:metricconverter><span lang=DE<br />
style='mso-ansi-language:DE'> MgSO<sub>4</sub> (<span class=SpellE>Invitrogen</span>)<br />
<o:p></o:p></span></p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;<br />
mso-border-right-alt:1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'>1.0 <span<br />
class=SpellE>&#956;L</span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:5'><br />
<td width=196 valign=top style='width:147.15pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>Primer 1 </p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;<br />
mso-border-right-alt:1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'>3.0 <span<br />
class=SpellE>&#956;L</span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:6'><br />
<td width=196 valign=top style='width:147.15pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>Primer 2 </p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;<br />
mso-border-right-alt:1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'>3.0 <span<br />
class=SpellE>&#956;L</span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:7'><br />
<td width=196 valign=top style='width:147.15pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal style='line-height:normal'><span class=SpellE>Pfx</span> <span<br />
class=SpellE>polymerase</span> (<span class=SpellE>Invitrogen</span>)</p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;<br />
mso-border-right-alt:1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'>0.6 <span<br />
class=SpellE>&#956;L</span> </p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:8'><br />
<td width=196 valign=top style='width:147.15pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>DNA <span class=SpellE>template</span></p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;<br />
mso-border-right-alt:1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'>1.0 <span<br />
class=SpellE>&#956;L</span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:9;mso-yfti-lastrow:yes'><br />
<td width=196 valign=top style='width:147.15pt;border:solid windowtext 1.5pt;<br />
border-top:none;mso-border-top-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>H<sub>2</sub>O </p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'>29.9 <span<br />
class=SpellE>&#956;L</span></p><br />
</td><br />
</tr><br />
</table><br />
<br/><br />
<p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>PCR program pFX : <br />
</span></p><br />
<br />
<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0 width=414<br />
style='width:310.55pt;margin-left:18.0pt;border-collapse:collapse;border:none;<br />
mso-border-alt:solid windowtext .5pt;mso-yfti-tbllook:480;mso-padding-alt:<br />
0cm 5.4pt 0cm 5.4pt;mso-border-insideh:.5pt solid windowtext;mso-border-insidev:<br />
.5pt solid windowtext'><br />
<tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'><br />
<td width=137 valign=top style='width:102.65pt;border:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><b>Step<o:p></o:p></b></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:solid windowtext 1.5pt;<br />
border-left:none;border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.0pt;<br />
mso-border-left-alt:solid windowtext 1.5pt;mso-border-alt:solid windowtext 1.5pt;<br />
mso-border-right-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE><b>Annealing</b></span><b><o:p></o:p></b></p><br />
<p class=MsoNormal><span class=SpellE><b>Temperature</b></span><b><o:p></o:p></b></p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:solid windowtext 1.5pt;<br />
border-left:none;border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.0pt;<br />
mso-border-left-alt:solid windowtext .5pt;mso-border-top-alt:1.5pt;<br />
mso-border-left-alt:.5pt;mso-border-bottom-alt:1.5pt;mso-border-right-alt:<br />
.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><b>Time, <o:p></o:p></b></p><br />
<p class=MsoNormal><b>min:sec<o:p></o:p></b></p><br />
</td><br />
<td width=91 valign=top style='width:68.0pt;border:solid windowtext 1.5pt;<br />
border-left:none;mso-border-left-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE><b>Number</b></span><b> of <span<br />
class=SpellE>cycles</span><o:p></o:p></b></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:1'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext 1.5pt;mso-border-alt:<br />
solid windowtext 1.5pt;mso-border-bottom-alt:solid windowtext .5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Initial</span> <span class=SpellE>denaturation</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext 1.5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:1.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:<br />
.5pt;mso-border-right-alt:.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>95 °C</p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext 1.5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-top-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>2:00</p><br />
</td><br />
<td width=91 rowspan=3 valign=top style='width:68.0pt;border-top:none;<br />
border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext 1.5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-top-alt:1.5pt;mso-border-left-alt:.5pt;mso-border-bottom-alt:.5pt;<br />
mso-border-right-alt:1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<p class=MsoNormal>1</p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:2'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Annealing</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span style='mso-spacerun:yes'> </span>X °C *</p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>1:00</p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:3'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Extension</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><st1:metricconverter ProductID="68&#65456;C" w:st="on">68°C</st1:metricconverter></p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>1:00-2:00 </p><br />
<p class=MsoNormal>(1 min/<span class=SpellE>kb</span>)</p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:4;page-break-inside:avoid'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Denaturation</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><st1:metricconverter ProductID="95 &#65456;C" w:st="on">95<br />
°C</st1:metricconverter></p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>1:00</p><br />
</td><br />
<td width=91 rowspan=3 valign=top style='width:68.0pt;border-top:none;<br />
border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-right-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<p class=MTDisplayEquation style='tab-stops:35.4pt'><span lang=EN-US<br />
style='font-size:11.0pt;mso-bidi-font-size:12.0pt;font-family:"Calibri","sans-serif"'>25<o:p></o:p></span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:5;page-break-inside:avoid'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Annealing</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span style='mso-spacerun:yes'> </span>X °C *</p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>1:00</p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:6;page-break-inside:avoid'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Extension</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><st1:metricconverter ProductID="68 &#65456;C" w:st="on">68<br />
°C</st1:metricconverter></p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>1:00-2:00 </p><br />
<p class=MsoNormal>(1 min/<span class=SpellE>kb</span>)</p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:7;mso-yfti-lastrow:yes;page-break-inside:avoid'><br />
<td width=137 valign=top style='width:102.65pt;border:solid windowtext 1.5pt;<br />
border-top:none;mso-border-top-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Final</span> <span class=SpellE>Extension</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:1.5pt;<br />
mso-border-right-alt:.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><st1:metricconverter ProductID="68 &#65456;C" w:st="on">68<br />
°C</st1:metricconverter></p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-bottom-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>10:00</p><br />
</td><br />
<td width=91 valign=top style='width:68.0pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>1</p><br />
</td><br />
</tr><br />
</table><br />
<br/><br />
<br />
<p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>For <span<br />
class=SpellE>Taq</span> PCR the reaction mixture is:<o:p></o:p></span></p><br />
<br />
<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0<br />
style='margin-left:18.0pt;border-collapse:collapse;border:none;mso-border-alt:<br />
solid windowtext .5pt;mso-yfti-tbllook:480;mso-padding-alt:0cm 5.4pt 0cm 5.4pt;<br />
mso-border-insideh:.5pt solid windowtext;mso-border-insidev:.5pt solid windowtext'><br />
<tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'><br />
<td width=196 valign=top style='width:147.15pt;border:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><b>Component<o:p></o:p></b></p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border:solid windowtext 1.5pt;<br />
border-left:none;mso-border-left-alt:solid windowtext 1.5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><b>Sample<o:p></o:p></b></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:1'><br />
<td width=196 valign=top style='width:147.15pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext 1.5pt;mso-border-alt:<br />
solid windowtext 1.5pt;mso-border-bottom-alt:solid windowtext .5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE><span lang=EN-US style='mso-ansi-language:<br />
EN-US'>Taq</span></span><span lang=EN-US style='mso-ansi-language:EN-US'> PCR<br />
Master Mix (<span class=SpellE>Qiagen</span><o:p></o:p></span></p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext 1.5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext 1.5pt;mso-border-bottom-alt:solid windowtext .5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'>12.5 <span<br />
class=SpellE>&#956;L</span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:2'><br />
<td width=196 valign=top style='width:147.15pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>Primer 1</p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;<br />
mso-border-right-alt:1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'><span lang=EN-US<br />
style='mso-ansi-language:EN-US'>1.5 </span>&#956;<span lang=EN-US<br />
style='mso-ansi-language:EN-US'>L<o:p></o:p></span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:3'><br />
<td width=196 valign=top style='width:147.15pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>Primer 2<o:p></o:p></span></p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;<br />
mso-border-right-alt:1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'><span lang=EN-US<br />
style='mso-ansi-language:EN-US'>1.5 </span>&#956;<span lang=EN-US<br />
style='mso-ansi-language:EN-US'>L<o:p></o:p></span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:4'><br />
<td width=196 valign=top style='width:147.15pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>Template<br />
DNA (or pick a colony)<o:p></o:p></span></p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;<br />
mso-border-right-alt:1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'><span lang=EN-US<br />
style='mso-ansi-language:EN-US'>1 <span class=SpellE>&#956;L</span><o:p></o:p></span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:5;mso-yfti-lastrow:yes'><br />
<td width=196 valign=top style='width:147.15pt;border:solid windowtext 1.5pt;<br />
border-top:none;mso-border-top-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>H<sub>2</sub>O<br />
<o:p></o:p></span></p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'><span lang=EN-US<br />
style='mso-ansi-language:EN-US'>8.5 </span>&#956;<span lang=EN-US<br />
style='mso-ansi-language:EN-US'>L<o:p></o:p></span></p><br />
</td><br />
</tr><br />
</table><br />
<br />
<br/><br />
<br />
<p class=MsoNormal>PCR program <span class=SpellE>Taq</span> <span<br />
class=SpellE>polymerase</span><span style='mso-bidi-font-weight:bold'>:</span></p><br />
<br />
<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0 width=414<br />
style='width:310.55pt;margin-left:18.0pt;border-collapse:collapse;border:none;<br />
mso-border-alt:solid windowtext .5pt;mso-yfti-tbllook:480;mso-padding-alt:<br />
0cm 5.4pt 0cm 5.4pt;mso-border-insideh:.5pt solid windowtext;mso-border-insidev:<br />
.5pt solid windowtext'><br />
<tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'><br />
<td width=137 valign=top style='width:102.65pt;border:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><b>Step<o:p></o:p></b></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:solid windowtext 1.5pt;<br />
border-left:none;border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.0pt;<br />
mso-border-left-alt:solid windowtext 1.5pt;mso-border-alt:solid windowtext 1.5pt;<br />
mso-border-right-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE><b>Annealing</b></span><b><o:p></o:p></b></p><br />
<p class=MsoNormal><span class=SpellE><b>Temperature</b></span><b><o:p></o:p></b></p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:solid windowtext 1.5pt;<br />
border-left:none;border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.0pt;<br />
mso-border-left-alt:solid windowtext .5pt;mso-border-top-alt:1.5pt;<br />
mso-border-left-alt:.5pt;mso-border-bottom-alt:1.5pt;mso-border-right-alt:<br />
.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><b>Time, <o:p></o:p></b></p><br />
<p class=MsoNormal><b>min:sec<o:p></o:p></b></p><br />
</td><br />
<td width=91 valign=top style='width:68.0pt;border:solid windowtext 1.5pt;<br />
border-left:none;mso-border-left-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE><b>Number</b></span><b> of <span<br />
class=SpellE>cycles</span><o:p></o:p></b></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:1'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext 1.5pt;mso-border-alt:<br />
solid windowtext 1.5pt;mso-border-bottom-alt:solid windowtext .5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Initial</span> <span class=SpellE>denaturation</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext 1.5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:1.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:<br />
.5pt;mso-border-right-alt:.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>94 °C</p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext 1.5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-top-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>10:00</p><br />
</td><br />
<td width=91 rowspan=3 valign=top style='width:68.0pt;border-top:none;<br />
border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext 1.5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-top-alt:1.5pt;mso-border-left-alt:.5pt;mso-border-bottom-alt:.5pt;<br />
mso-border-right-alt:1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<p class=MsoNormal>1</p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:2'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Annealing</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span style='mso-spacerun:yes'> </span>X °C *</p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>0:45</p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:3'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Extension</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>72°C</p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>1:00-2:00 </p><br />
<p class=MsoNormal>(1 min/<span class=SpellE>kb</span>)</p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:4;page-break-inside:avoid'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Denaturation</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><st1:metricconverter ProductID="95 &#65456;C" w:st="on">95<br />
°C</st1:metricconverter></p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>1:00</p><br />
</td><br />
<td width=91 rowspan=3 valign=top style='width:68.0pt;border-top:none;<br />
border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-right-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<p class=MTDisplayEquation style='tab-stops:35.4pt'><span lang=EN-US<br />
style='font-size:11.0pt;mso-bidi-font-size:12.0pt;font-family:"Calibri","sans-serif"'>25<o:p></o:p></span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:5;page-break-inside:avoid'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Annealing</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span style='mso-spacerun:yes'> </span>X °C *</p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>1:00</p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:6;page-break-inside:avoid'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Extension</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>72 °C</p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>1:00-2:00 </p><br />
<p class=MsoNormal>(1 min/<span class=SpellE>kb</span>)</p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:7;mso-yfti-lastrow:yes;page-break-inside:avoid'><br />
<td width=137 valign=top style='width:102.65pt;border:solid windowtext 1.5pt;<br />
border-top:none;mso-border-top-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Final</span> <span class=SpellE>Extension</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:1.5pt;<br />
mso-border-right-alt:.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>72 °C</p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-bottom-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>10:00</p><br />
</td><br />
<td width=91 valign=top style='width:68.0pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>1</p><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<br/><br />
<br />
<p>*Annealing temperature is very dependent on primer. Optimal temperature: 3x G/C + 2x A/T </p><br />
<br />
<br />
<br/><br />
<br />
<a name="P8"> <h1>Gel Extraction</h1></a><br />
<br/><br />
This protocol is based on QIAGEN® Gel Extraction Handbook.<br />
<br/><br/><br />
<i>Materials:</i><br />
<br/><br />
<ul><li>QIAquick columns</li><li><br />
buffer QG</li><li><br />
buffer PE</li><li><br />
isopropanol</li><li><br />
milliQ</li><li><br />
microcentrifuge</li><li><br />
heat block at 50 °C</li><br />
</ul><br />
<br/><br />
<i>Protocol:</i><br />
<ol><br />
<li>Excise the DNA fragment from the agarose gel with a clean, sharp scalpel. Minimize the size of the gel slice by removing extra agarose. <br />
</li><br />
<li><br />
Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg ~ 100 µL). For >2% agarose gels, add 6 volumes of Buffer QG. The maximum amount of gel slice per QIAquick column is 400 mg; for gel slices >400 mg use more than one QIAquick column. <br />
</li><br />
<li><br />
Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2–3 min during the incubation. <br/><br />
IMPORTANT: Solubilize agarose completely. For >2% gels, increase incubation time. <br />
</li><br />
<li><br />
After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 µL of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow. The adsorption of DNA to the QIAquick membrane is efficient only at pH <7.5. Buffer QG contains a pH indicator which is yellow at pH <7.5 and orange or violet at higher pH, allowing easy determination of the optimal pH for DNA binding. <br />
</li><br />
<li><br />
Add 1 gel volume of isopropanol to the sample and mix. This step increases the yield of DNA fragments <500 bp and >4 kb. For DNA fragments between 500 bp and 4 kb, addition of isopropanol has no effect on yield. Do not centrifuge the sample at this stage. <br />
</li><br />
<li><br />
Place a QIAquick spin column in a provided 2 ml collection tube. <br />
</li><br />
<li><br />
To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 min. <br />
The maximum volume of the column reservoir is 800 µL. For sample volumes of more than 800 µL, simply load and spin again. <br />
</li><br />
<li><br />
Discard flow-through and place QIAquick column back in the same collection tube. <br />
</li><br />
<li><br />
To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min. <br />
</li><br />
<li><br />
Discard the flow-through and centrifuge the QIAquick column for an additional 1 min at 10,000 x g (~13,000 rpm). <br />
</li><br />
<li><br />
IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation. <br />
</li><br />
<li><br />
Place QIAquick column into a clean 1.5 ml microcentrifuge tube. <br />
</li><br />
<li><br />
To elute DNA, add 50 µL of Buffer EB (10 mM Tris•Cl, pH 8.5) or H2O to the center of the QIAquick membrane and centrifuge the column for 1 min at maximum speed. Alternatively, for increased DNA concentration, add 30 µl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min. <br/><br />
Important: Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA. The average eluate volume is 48 µL from 50 µL elution buffer volume, and 28 µL from 30 µL. Elution efficiency is dependent on pH. The maximum elution efficiency is achieved between pH 7.0 and 8.5. When using water, make sure that the pH value is within this range, and store DNA at –20°C as DNA may degrade in the absence of a buffering agent. The purified DNA can also be eluted in TE (10 mM Tris•Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.<br />
</li><br />
</ol><br />
<br />
<br/><br/><br />
<br />
<br />
<a name="P11"> <h1>Immunofluorescence Staining</h1></a><br />
<br/><br />
<div class=WordSection1><br />
<br />
<p class=MsoNoSpacing><span lang=EN-US>This protocol is for immunofluorescence staining<br />
of yeast to prepare it for Flow <span class=SpellE>Cytometry</span> or fluorescence<br />
microscopy, and staining the target of the conjugated antibody of choice. The<br />
TU Delft <span class=SpellE>iGEM</span> 2012 team used a <a<br />
href="http://www.cellsignal.com/products/3916.html"><span class=SpellE>flagtag</span><br />
antibody.</a> <span class=GramE>The</span> protocol of the supplier was<br />
improved for yeast staining. <b style='mso-bidi-font-weight:normal'>IMPORTANT:</b>&nbsp;Please<br />
refer to the&nbsp;<b style='mso-bidi-font-weight:normal'>APPLICATIONS</b><span<br />
class=GramE>&nbsp; section</span> on the front page of the datasheet of the<br />
antibody product to determine if this product is validated and approved for use<br />
on cultured cell lines (IF-IC).</span></p><br />
<br />
<p class=MsoNoSpacing><span lang=EN-US style='mso-fareast-language:NL'><o:p>&nbsp;</o:p></span></p><br />
<br />
<p class=MsoNoSpacing><span lang=EN-US style='mso-bidi-font-family:Calibri;<br />
mso-bidi-theme-font:minor-latin;mso-fareast-language:NL'>Materials:<o:p></o:p></span></p><br />
<br />
<p class=MsoNoSpacing><span lang=EN-US style='mso-bidi-font-family:Calibri;<br />
mso-bidi-theme-font:minor-latin;mso-fareast-language:NL'><o:p>&nbsp;</o:p></span></p><br />
<br />
<p class=MsoNoSpacing style='margin-left:36.0pt;text-indent:-18.0pt;mso-list:<br />
l3 level1 lfo13'><![if !supportLists]><span lang=EN-US style='font-family:Symbol;<br />
mso-fareast-font-family:Symbol;mso-bidi-font-family:Symbol;mso-fareast-language:<br />
NL'><span style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span lang=EN-US style='mso-fareast-language:<br />
NL'><a href="http://openwetware.org/wiki/PBS">Phosphate Buffered Saline (PBS)</a>,<br />
adjust pH to 8.0.<o:p></o:p></span></p><br />
<br />
<p class=MsoNoSpacing style='margin-left:36.0pt;text-indent:-18.0pt;mso-list:<br />
l3 level1 lfo13'><![if !supportLists]><span lang=EN-US style='font-family:Symbol;<br />
mso-fareast-font-family:Symbol;mso-bidi-font-family:Symbol;mso-fareast-language:<br />
NL'><span style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span lang=EN-US style='mso-fareast-language:<br />
NL'>Formaldehyde, 16%, <b style='mso-bidi-font-weight:normal'>methanol free</b><o:p></o:p></span></p><br />
<br />
<p class=MsoNoSpacing style='margin-left:36.0pt;text-indent:-18.0pt;mso-list:<br />
l3 level1 lfo13'><![if !supportLists]><span lang=EN-US style='font-family:Symbol;<br />
mso-fareast-font-family:Symbol;mso-bidi-font-family:Symbol;mso-fareast-language:<br />
NL'><span style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span lang=EN-US style='mso-fareast-language:<br />
NL'>Blocking Buffer:&nbsp;(1X PBS / 1 <span class=SpellE>wt</span>% BSA / 0.3%<br />
(v/v) Triton X-100)<o:p></o:p></span></p><br />
<br />
<p class=MsoNoSpacing style='margin-left:36.0pt;text-indent:-18.0pt;mso-list:<br />
l3 level1 lfo13'><![if !supportLists]><span lang=EN-US style='font-family:Symbol;<br />
mso-fareast-font-family:Symbol;mso-bidi-font-family:Symbol;mso-fareast-language:<br />
NL'><span style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span lang=EN-US style='mso-fareast-language:<br />
NL'>Antibody Dilution Buffer&nbsp;(1X PBS / 1 <span class=SpellE>wt</span>% BSA<br />
/ 0.3% (v/v) Triton X-100)<o:p></o:p></span></p><br />
<br />
<p class=MsoNoSpacing style='margin-left:36.0pt;text-indent:-18.0pt;mso-list:<br />
l3 level1 lfo13'><![if !supportLists]><span lang=EN-US style='font-family:Symbol;<br />
mso-fareast-font-family:Symbol;mso-bidi-font-family:Symbol;mso-fareast-language:<br />
NL'><span style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span lang=EN-US style='mso-fareast-language:<br />
NL'>Primary conjugated antibody.<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:<br />
22.2pt;background:white'><b><span style='mso-fareast-font-family:"Times New Roman";<br />
mso-bidi-font-family:Calibri;mso-bidi-theme-font:minor-latin;color:#2B6EAE;<br />
mso-ansi-language:NL;mso-fareast-language:NL'><o:p>&nbsp;</o:p></span></b></p><br />
<br />
<p class=MsoNoSpacing><span lang=EN-US style='mso-fareast-language:NL'>Protocol:<o:p></o:p></span></p><br />
<br />
<p class=MsoNoSpacing><span lang=EN-US style='mso-fareast-language:NL'><o:p>&nbsp;</o:p></span></p><br />
<br />
<p class=MsoNoSpacing><span lang=EN-US style='mso-fareast-language:NL'>The<br />
desired culture of cell is taken for staining and the liquid is aspirated by<br />
spinning it down in appropriate tubing <span class=GramE>( e.g</span>. standard<br />
<span class=SpellE>Eppendorf</span> tubes )<span style='mso-spacerun:yes'> <br />
</span>and decanting or pipetting the supernatant.<span<br />
style='mso-spacerun:yes'> </span>Afterwards the container is inspected for a<br />
cell pellet of acceptable size, meaning it must be easy to spot in the<br />
container. After this the fixing of the cells can start:<o:p></o:p></span></p><br />
<br />
<p class=MsoNoSpacing><b><span lang=EN-US style='color:#2B6EAE;mso-fareast-language:<br />
NL'><o:p>&nbsp;</o:p></span></b></p><br />
<br />
<p class=MsoNoSpacing style='margin-left:36.0pt;text-indent:-18.0pt;mso-list:<br />
l9 level1 lfo14'><![if !supportLists]><span lang=EN-US style='mso-bidi-font-family:<br />
Calibri;mso-bidi-theme-font:minor-latin;mso-fareast-language:NL'><span<br />
style='mso-list:Ignore'>1.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span lang=EN-US style='mso-fareast-language:<br />
NL'>Cover cells to a depth of 10–15 mm with 4% formaldehyde in PBS and <span<br />
class=SpellE>resuspend</span> cells.<br/><br />
<b>NOTE:</b>&nbsp;Formaldehyde is toxic, use only in fume hood.<o:p></o:p></span></p><br />
<br />
<p class=MsoNoSpacing style='margin-left:36.0pt;text-indent:-18.0pt;mso-list:<br />
l9 level1 lfo14'><![if !supportLists]><span lang=EN-US style='mso-bidi-font-family:<br />
Calibri;mso-bidi-theme-font:minor-latin;mso-fareast-language:NL'><span<br />
style='mso-list:Ignore'>2.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span lang=EN-US style='mso-fareast-language:<br />
NL'>Allow cells to fix for 15 minutes at room temperature.<o:p></o:p></span></p><br />
<br />
<p class=MsoNoSpacing style='margin-left:36.0pt;text-indent:-18.0pt;mso-list:<br />
l9 level1 lfo14'><![if !supportLists]><span lang=EN-US style='mso-bidi-font-family:<br />
Calibri;mso-bidi-theme-font:minor-latin;mso-fareast-language:NL'><span<br />
style='mso-list:Ignore'>3.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span lang=EN-US style='mso-fareast-language:<br />
NL'>Aspirate fixative and rinse three times in PBS for 5 minutes each before<br />
proceeding to the staining section. <span class=GramE>( Use</span> the same<br />
aspirating techniques as above: Spin, Aspirate, add, <span class=SpellE>resuspend</span>.<br />
)<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;<br />
margin-left:48.0pt;margin-bottom:.0001pt;line-height:22.2pt;background:white'><span<br />
lang=EN-US style='mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:<br />
Calibri;mso-bidi-theme-font:minor-latin;color:black;mso-fareast-language:NL'><o:p>&nbsp;</o:p></span></p><br />
<br />
<p class=MsoNoSpacing><span lang=EN-US style='mso-fareast-language:NL'>All subsequent<br />
incubations should be carried out at room temperature unless otherwise noted in<br />
a humid light-tight box or covered dish/plate to prevent drying and <span<br />
class=SpellE>fluorochrome</span> fading.<o:p></o:p></span></p><br />
<br />
<p><ol><br />
<br />
<li> Block specimen in Blocking Buffer for 60 minutes.</li><br />
<li> While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer. ( A Cell Signaling technology antibody was used with this protocol )</li><br />
<li> Aspirate blocking solution, apply diluted primary antibody.</li><br />
<li> Incubate overnight at 4°C.</li><br />
<li> Rinse three times in PBS for 5 minutes each.<br />
NOTE: Because one is using primary antibodies directly conjugated with ( Alexa Fluor® ) fluorochromes, secondary antibodies are not needed.</li><br />
<li> For best results, examine specimens immediately using appropriate excitation wavelength. For long-term storage, store slides flat at 4°C protected from light.</li><br />
</ol></p><br />
<br />
<br />
</div><br />
<br />
<br />
<br/><br />
<a name="P12"><h1>DNA Staining</h1></a><br/><br/><br />
<i>Materials</i><br/><br />
<ul><li>Your favorite yeast strain</li><br />
<li>Vybrant DyeCycle Orange from invitrogen</li><br />
<li>PBS</li><br />
<li>37°C heat block</li><br />
</ul><br/><br/><br />
<br />
<i>Protocol</i><br/><br />
<ol><li>Centrifuge cells on a table top centrifuge for 10 s on 13.000 rpm </li><br />
<li>Take of supernatant and add PBS</li><br />
<li>Add 2 µL/mL Vybrant DyeCycle Orange and vortex until homogenuous solution</li><br />
<li>Keep for 30 minutes on 37°C and the cells are stained.</li><br/><br/><br />
</ol><br />
<br />
<a name="P13"><h1>Flow cytometry to look at DNA content</h1></a><br/><br />
<i>Materials</i><br/><br />
<ul><li>Yeast culture</li><br />
<li>Vybrant® DyeCycleTM Orange stain</li><br />
<li>Ligand</li><br />
<li>Flow cytometry instrument</li><br/><br />
</ul><br />
<i>Protocol</i><br/><br />
<ol><br />
<li>Grow yeast cells at 30C overnight and re-inocculate in 10 mL medium the next morning</li><br />
<li>If cells are in exponential growth phase add the ligand (also keep a control without ligand)</li><br />
<li>After incubation stain the cells according to protocol</li><br />
<li>Transfer the stained cells in a flow cytometry tube</li><br />
<li>Analyze the samples on a flow cytometer using 488 nm excitation or 532 nm excitation and orange emission</li><br />
<li>After each measurement flush the tube with PBS buffer</li><br />
</ol><br />
<br/><br />
<br/><br />
<a name="P14"><h1>Flow cytometry to look at EGFP expression</h1></a><br/><br />
<br />
<br />
<i>Materials</i><br/><br />
<ul><br />
<li>Yeast culture</li><br />
<li>Ligand</li><br />
<li>Flow cytometry instrument</li><br />
</ul><br />
<br/><br />
<i>Protocol</i><br/><br />
<ol><li>Grow yeast cells at 30C overnight and re-inocculate in 10 mL medium the next morning</li><br />
<li>If cells are in exponential growth phase add the ligand (also keep a control without ligand)</li><br />
<li>Transfer the cells in a flow cytometry tube</li><br />
<li>Analyze the samples on a flow cytometer using 488 nm excitation and 509 nm emission</li><br />
<li>After each measurement flush the tube with PBS buffer</li><br />
</ol><br />
<br/><br />
<br/><br />
<a name="P15"><h1>Fluorimeter experiment</h1></a><br/><br/><br />
<i>Materials</i><br/><br />
<ul><br />
<li>Yeast culture</li><br />
<li>Ligand</li><br />
<li>Fluorimeter plate reader</li><br />
<li>96 well plate</li><br/><br />
</ul><br />
<i>Protocol</i><br />
<br />
<br/><br />
<ol><li>Grow yeast cells at 30C overnight and re-inocculate the next morning</li><br />
<li>Measure after approx. 3h hours the OD600. Dilute the cells with medium until an OD of approx. 0.1</li><br />
<li>Transfer the cells to the 96 well plate. Don't forget to have a well with only medium.</li><br />
<li>Add the ligand.</li><br />
<li>Analyze the samples in the fluorimeter that is kept at 30C. Use filters with 488 nm excitation and 509 nm emission. At the same time measure the OD600.</li><br />
<li>Calculate the fluoresce per biomass by dewing the fluorescence by the OD600 values</li><br />
</ol><br><br />
<br />
<a name="P17"><h1>Growth rate experiment</h1></a><br/><br/><br />
<i>Materials</i><br/><br />
<ul><br />
<li>Yeast cultures (wt with and without alpha feromones and far1KO with and without alpha feromones)</li><br />
<li>Alpha feromones</li><br />
<li>Spectrophotometer</li><br />
<li>30C incubator</li><br/><br />
</ul><br />
<i>Protocol</i><br />
<br />
<br/><br />
<ol><li>Grow yeast cells at 30C overnight and re-inocculate 20 ml of medium with 500µl of grown cells the next morning</li><br />
<li>Measure OD600 every hour. </li><br />
<li>Add alpha feromones after 3hr</li><br />
<li>Measure OD600every hour, dilute if needed.</li><br />
</ol><br><br />
<br />
<a name="P18"><h1>Smell activity tests with petri dishes</h1></a><br/><br/><br />
<i>Materials</i><br/><br />
<br />
<ul><br />
<br />
<li>4 x Two yeast cultures with each having six yeast-strains</li><br />
<br />
<li>Ligands </li><br />
<br />
Water 2 x<br />
<br />
Alpha pheromone 1000x diluted 2 x<br />
<br />
Isoamyl acetate 99%<br />
<br />
Methyl phenylacete 99%<br />
<br />
Niacin 0.336 g/100 ml<br />
<br />
Methyl nicotinate 10 mM<br />
<br />
<li>Typhoon fluorescence scanner</li><br />
<br />
<li>8 Resealable (hard) plastic boxes of 10x10x10 cm.</li><br/><br />
<br />
</ul><br />
<br />
<br/><br />
<br />
<i>Protocol</i><br />
<br />
<br/><br />
<br />
<ol><li>Grow yeast cells at 30C for two days.</li><br />
<br />
<li>Analyze the samples in the Typhoon fluorescence scanner before starting experiment.</li><br />
<br />
<li>Pore approx. 25 ml of every ligand (except alpha pheromone) in the box after cleaning it with alcohol / autoclaving if possible. </li><br />
<br />
<li>Place two sided tape on the inwards part of the boxlid. Gently but firmly push the petri dish on it. Remove the lid of the dish and put the dish upside down in the box. For alpha pheromone; add solution directly on all yeast strains.</li><br />
<br />
<li>After 3.5 hours remove the dish from the lid.</li><br />
<br />
<li>Analyze the samples in the Typhoon fluorescence scanner. Use filters with 488 nm excitation and 509 nm emission. Set PMT to 300. Orientation of dish to Platen.</li><br />
<br />
</ol><br><br><br />
<br />
<br />
<a name="P16"><h1>Tips&Tricks for working with yeast</h1></a><br />
<br />
<p>Since this is the first time that TU Delfts iGEM team is working with yeast, we faced a lot of small yeast-related 'challenges'. With this page we want to inform you about the basics and the pitfalls of working with yeast. </p><br />
<h3>Basics</h3><br />
<p>Yeast, <i>Saccharomyces cerevisiae</i>, is a simple, unicellular eukaryotic organism. This organism has been used for fermentation and baking for over 4000 years and it is probably the oldest domesticated micro-organism in the human history. An important one too, can you imagine a life without bread and beer? (Even if you can imagine it, it would most definitely be less fun!) <br />
Nowadays the whole genomic sequence of <i>Saccharomyces cerevisiae</i> is known and a lot of genomic tools are available.</p><br />
<h3>Auxotrophy</h3><br />
<p> The main advantage from an engineering perspective is that yeast has Auxotrophic markers. In specific strains genes are knocked out which synthesize essential enzymes in the amino acid synthesis routes. By complementing these deficiencies by adding the necessary gene on your DNA this provides a nice selection procedure. <br />
<h3> PRS huttle vectors</h3> <br />
<p>The name pRS415 gives an indication on the presence of a CEN/ARS replication origin. 0 means yeast<br />
integrative plasmid, 1 means that it also can be used to maintain the plasmid in circular<br />
form.<br />
pRS415 Gives an indication of the auxotrophic marker used.<br />
pRS415 Version number… not really different.<br/><br />
<h3>Chromosomal integration</h3><br />
<p>We encountered a lot of problems with plasmids. Because we wanted our constructs to be universal<br />
(with the idea to make it suitable for ‘fast checking’) we tried maintaining a plasmid. As it turned<br />
out, yeast cells are not eager to maintain a plasmid and with our construct we suspect homologous<br />
recombination occurred. After transformation, a PCR on the transformed plasmid, obtained by<br />
isolation, showed two bands instead of the suspected single band, one being ! Integration of the<br />
plasmid is therefore advised! Checking of this can be quite gruesome optimizing the necessary PCR<br />
reactions on your transformed yeast colonies. Chromosomal isolation can therefore improve the<br />
steps. </p><br/><br />
<h3>Knock-out strains</h3> <br />
<p>European iGEM teams have the advantage to have <a href="http://web.uni-<br />
frankfurt.de/fb15/mikro/euroscarf">Euroscarf</a> available to order strains with knocked out ORFs. The typical<br />
nomenclature is also explained here: <a href="http://web.uni-frankfurt.de/fb15/mikro/euroscarf/<br />
stra_des.html">Euroscarf explanation</a>.</p><br/><br />
<br />
<br />
<br />
<br />
</div></div><br />
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<a href='https://2012.igem.org/Main_Page' target="_blank"><div id='logo_igem2'></div><a/></body></html></div>MarkWeijershttp://2012.igem.org/Team:TU-Delft/protocolsTeam:TU-Delft/protocols2012-10-27T03:06:57Z<p>MarkWeijers: </p>
<hr />
<div>For a new page in our wiki <br />
{{:Team:TUDelft/Scripts}}{{:Team:TUDelft/CSS}}{{:Team:TUDelft/menu}}<br />
<html><br />
<head><title>Your title</title></head><br />
<body><br />
<div style="height:70px; width:100%;"></div><br />
<div id="logo_ed"><a href="https://2012.igem.org/Team:TU-Delft" 'onfocus=this.blur()'><br />
<img src="https://static.igem.org/mediawiki/igem.org/e/e2/Igemlogo.png" border="0" width="100" height="100"/></a></div><br />
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<br />
<br />
<br />
<h1>Protocols</h1> <br />
<br />
<a href="#P10"> Media</a><br/><br />
<a href="#P9"> Yeast Transformation</a><br/><br />
<a href="#P1"> Transforming One Shot® Mach1™ competent cells </a><br/><br />
<a href="#P2"> Mini-prep plasmid isolation for E. coli and S. cerevisae </a><br/><br />
<a href="#P3"> Freezing cell stocks </a><br/><br />
<a href="#P4"> Restriction enzyme digestion </a><br/><br />
<a href="#P5"> Ligation </a><br/><br />
<a href="#P6"> Running DNA Gel </a><br/><br />
<a href="#P7"> PCR </a><br/><br />
<a href="#P8">Gel Extraction</a><br/><br />
<a href="#P11">Immunofluorescence Staining</a><br/><br />
<a href="#P12">DNA Staining</a><br/><br />
<a href="#P13">Flow cytometry to look at DNA content</a><br/><br />
<a href="#P14">Flow cytometry to look at EGFP expression</a><br/><br />
<a href="#P15">Fluorimeter experiment</a><br/><br />
<a href="#P17">Growth curve experiment</a><br/><br />
<a href="#P18">Smell activity tests with petri dishes</a><br/><br />
<a href="#P16">Tips&Tricks for working with yeast</a><br/><br/><br />
<br />
<a name="P10"><h1>Media</h1></a><br />
<br/><br />
<i>DO (agar)</i><br />
<br/>For BY4741 yeast strain add to mineral media:<br/>HIS: 125 mg/liter<br/>LEU 500 mg/liter<br/>MET 100 mg/liter<br/>URA 150 mg/liter<br/>except for one which you want to select on with you auxotrophic marker.<br/><br/>Also add:<br/>2% Sucrose (w/v)<br/>2% Agarose<br/><br/>and autoclave<br/><br/><br />
<i>YPD (agar)</i><br />
<br/>http://openwetware.org/wiki/YPD <br/><br/><br />
<i>LB (agar) </i><br/><br />
<br/>http://openwetware.org/wiki/LB <br/><br/><br />
<br />
<a name="P9"><h1>Yeast Transformation</h1></a><br />
<br/><br />
<i>Materials</i><br/><br />
<ul><li>Yeast culture</li><br />
<li>single stranded carrier DNA (200 mg Salmon sperm DNA inTE)</li><br />
<li>Lithium Acetate (1.0 M)</li><br />
<li>Polyethylene Glycol 3350 (50% w/v)</li><br />
<li>DO media plates </li><br />
<li>Dropout (DO) media, lacking an amino acid of which an auxotrophic marker is added</li><br />
<li>Water bath 42 °C</li><br />
<li>Water bath 30 °C</li><br/><br/><br />
</ul><br />
<br />
<i>Protocol</i><br/><br />
<br />
<br />
<ol><li> Grow yeast culture overnight in 10 mL YPD on 30 °C</li><br />
<li> Put 0.5 mL – 1 mL into new flask with 20 mL YPD (for 2-4 transformations) and grow 4-5 hours until an OD of 0.8 is measured. The cells are now in exponential phase.</li><br />
<li> Spin the cells for 5 minutes on 3000 G and discard supernatant</li><br />
<li> Wash cells with water and spin down for 5 minutes on 3000 G</li><br />
<li> During centrifuging, make a transformation mix (TM): Per reaction add <br/><br />
<ul><li>240 µL 50% PEG</li><br />
<li>36 µL 1M Lithium Acetate solution</li><br />
<li>25 µL ssDNA, boiled for 5 minutes and then cooled on ice</li><br />
<li>49 µL water</li><br />
</ul><br/><br />
</li><br />
<li> Discard supernatant and resuspend in 0.2 mL 0.1 M Lithium Acetate solution</li><br />
<li> Do a quick centrifuge (10 s 13.000 rpm on a table top centrifuge), discard supernatant and add 80 µL 0.1 M Lithium Acetate solution. Vortex and do <u>not</u> pipette up and down</li><br />
<li>You should have ~100 µL of which 50 µL should be put in a fresh tube. This is you negative control</li><br />
<li>spin cells down again and add the mastermix</li><br />
<li>Add 1 µL target (100 ng – 1000 ng) DNA to the positive tube</li><br />
<li>Put in a 30 °C water bath for 30 minutes</li><br />
<li>Put it in a 42 °C water bath for 30 minutes</li><br />
<li>Do a quick centrifuge (10 s 13.000 rpm on a table top), remove supernatant. Add 0.2 mL water, pipet up and down and plate 150 µL and 50 µL on DO selective plates </li><br />
</ol><br/><br/><br />
<br />
<br />
<a name="P1"> <h1>Transforming One Shot® Mach1™ competent cells</h1></a><br/><br />
<br />
<br/><br />
<i>Materials:</i><br />
<br/><br />
<ul><br />
<li>- Competent cells</li><br />
<li> - SOC medium (warmed to room temperature)</li><br />
<li> - Plasmid DNA or DNA ligation mix</li><br />
<li> - LB agar plates containing 15-100 ?g/mL antibiotic of choice, pre-warmed to 37 °C </li><br />
<li> - water bath at 42 °C </li><br />
<li> - shaking incubator at 37 °C. </li><br />
</ul><br />
<br/><br />
<i>Protocol:</i><br />
<br/><br />
<ol><li>Add 50-100 ng DNA into a 20 ?L competent E.coli, and mix gently. Do not mix by pipetting up and down!</li><br />
<li>Incubate tube vial on ice for 30 minutes</li><br />
<li>Heat-shocks the cells for 30 seconds at 42 °C without shaking</li><br />
<li>Immediately transfer the tubes back to ice for 2 minutes</li><br />
<li>Add 250 ?L of room temperature LB medium</li><br />
<li>Cap tube tightly and shake tube horizontally (225 rpm) at 37 °C for 1 hour</li><br />
<li>Plate from each tube 100 ?L on an agar plate containing antibiotic. Spin tube, discard supernatant to leave no more than 100 ?L, vortex and plate on an agar plate</li><br />
<li>Incubate plates overnight at 37 °C</li><br />
</ol><br />
<br/><br />
<br />
<br />
<br />
<a name="P2"> <h1> Qiagen Mini-prep plasmid isolation for E. coli and S. cerevisae</h1></a><br />
<br/><br />
This protocol is based on QIAGEN® Plasmid Purification Handbook.<br/><br />
<i>Materials:</i><br/><br />
<ul><br />
<li>- bacterial or yeast culture</li><br />
<li>- Qiagen colums</li><br />
<li>- buffer P1 (100 mg/mL RNAse A, 50 mM Tris/HCl, 10 mM EDTA, pH 8.0) </li><br />
<li>- buffer P2 (200 mM NaOH, 1% SDS) </li><br />
<li>- buffer P3 (3 M KAc, pH 5.5) </li><br />
<li>- buffer PE </li><br />
<li>- milliQ pH 8.0</li><br />
<li>- centrifuge</li><br />
<li>- nanodrop</li><br />
<li>- for yeast plasmid isolation: zymolyase 5 U/µl</li></ul><br/><br />
<i> Protocol:</i><br/><br />
<ol><br />
<li>Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 2–5 mL LB medium containing the appropriate selective antibiotic or selective medium. Incubate for approximately 8 h at 37°C (bacteria) or 12 h at 30°C with vigorous shaking (approx. 300 rpm)</li><br />
<li>Harvest the 5 mL bacterial cells by centrifugation at 13,000 rpm for 1 min at 20°C (microcentrifuge tube). If you wish to stop the protocol and continue later, freeze the cell pellets at –20°C</li><br />
<li>For bacteria: Resuspend pelleted bacterial cells in 250 µL Buffer P1. Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet. <br/> For yeast: Resuspend cells in 250 µL Buffer P1 with 3 µL zymolyase and incubate 1 h at 37 °C</li><br />
<li>Add 250 µL Buffer P2 and mix thoroughly by inverting the tube 4–6 times. Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. 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><br />
<li>Add 350 µL Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer N3. Large culture volumes (e.g. =5 mL) may require inverting up to 10 times. The solution should become cloudy. </li><br />
<li>Incubate at -20 °C for 15 minutes. </li><br />
<li>Centrifuge for 10 min at 13,000 rpm in a table-top microcentrifuge. A compact white pellet will form. </li><br />
<li>Apply the supernatants from step 7 to the QIAprep spin column by decanting or pipetting. </li><br />
<li>Centrifuge for 30–60 seconds. Discard the flow-through. </li><br />
<li>Wash QIAprep spin column by adding 0.75 mL Buffer PE and centrifuging for 30–60 seconds. </li><br />
<li>Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer. <br />
<b>Important:</b> Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions. </li><br />
<li>Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 30 µL Buffer EB (10 mM Tris•Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 minute in 50 C stove and centrifuge for 1 minute to obtain DNA. </li><br />
<li>Measure DNA concentration on the Nanodrop</li><br />
</ol><br/><br />
<br />
<br />
<br />
<br />
<a name="P3"> <h1>Freezing cell stocks</h1></a><br />
<br/><br />
<i>Materials:</i> <br />
<ul><br />
<li>- bacterial culture </li><br />
<li>- Growth medium</li><br />
<li>- 80% glycerol</li><br />
<li>- centrifuge</li><br />
</ul><br />
<br/><br />
<br />
<i>Protocol:</i><br/><br />
<ol><br />
<li>Take 5 mL bacterial cells from the Erlenmeyer of a freshly grown culture and spin in a 15 mL tube for 10 minutes at 2.000 rpm (Eppendorf centrifuge)</li><br />
<li>Decant the supernatant without disturbing the pellet</li><br />
<li>Pipet on the pellet 0.5 ml of appropriate medium and 0.5 mL 80% glycerol and mix by vortexing and save in -80 °C freezer</li><br />
</ol><br />
<br/><br />
<br />
<br />
<br />
<br />
<a name="P4"> <h1> Restriction enzyme digestion </h1></a><br />
<br/><br />
<i>Materials:</i><br/><br />
<ul><br />
<li>- plasmid DNA or PCR product </li><br />
<li>- restriction enzymes (Roche and BioLabs) </li><br />
<li>- buffer (10x) </li><br />
<li>- H2O</li><br />
<li>water bath at 37 °C</li><br />
<li>- heat block or water bath at 65 °C</li></ul><br/><br />
<i>Protocol:</i><br/><br />
Digestions (cutting plasmid DNA) were performed at the appropriate temperature with the appropriate buffer in the appropriate concentration, according to the supplier. With double restriction, use bigger volume (~ 50 µL) and we found out that adding BSA altered performance greatly.<br/><br/><br />
<br/>Reaction for one sample:<br/><br />
DNA × µL (up to 1,0 µg)<br/><br />
Buffer (10×) × µL (1×))<br/><br />
Restriction enzymes × µL (5 units/µg DNA = 1 µL) )<br/><br />
H2O × µL<br/><br />
tot volume 20-25 µL<br />
<br/><br/><br />
Incubate for (at least) one hour at 37 °C. Inactivate the restriction endonucleases by heat, incubation at 65 °C for 10 minutes and centrifuge shortly.<br/><br />
<br/><br />
<i><u>Used Buffers:</u></i><br/><br />
Buffer H (Roche): 50 mM Tris-HCl, 1 M NaCl, 100 mM MgCl2, 10 mM DTE, pH 7.5 at 37 °C<br/><br />
Buffer M (Roche): 100 mM Tris-HCl, 500 mM NaCI, 100 mM MgCl2, 10 mM DTE, pH 7.5 at 37 °C<br/><br />
Buffer 1 (BioLabs): 10 mM Bis-Tris-Propane-HCl, 10 mM MgCl2, 1 mM DTE,pH 7.0 at 25°C<br/><br />
Buffer 2 (BioLabs): 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTE, pH 7.9 at 25°C<br/><br />
Buffer 3 (BioLabs): 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 1 mM DTE, pH 7.9 at 25°C<br/><br />
Buffer 4 (BioLabs): 50 mM CH3CO2K, 20 mM TAE, 10 mM Mg(CH3COO)2, 1 mM DTE, pH 7.9 at 25°C<br/><br/><br />
Note: some of the restriction enzymes of New England BioLabs required the addition of 100 µg/mL BSA)<br/><br />
<br />
<br><br />
<br />
<br />
<br />
<a name="P5"> <h1>Ligation</h1></a><br />
<br/><br />
<i>Materials:</i><br />
<ul><br />
<li>- digested plasmid DNA or PCR product</li><br />
<li>- T4 ligation buffer (10x) (Fermentas)</li><br />
<li>- T4 ligase (Fermentas)</li><br />
<li>- H2O</li><br />
<li>water bath at 16 °C</li><br />
</ul><br />
<br/><br />
<i>Protocol:</i><br />
Ligations (pasting plasmid DNA) were performed at the appropriate temperature with the appropriate buffer in the appropriate concentration, according to the supplier. We encountered different tactics for ligation. Usually it comes down to keeping it ~16 °C for at least 3 hours.<br />
<br/><br/><br />
<br />
Reaction for one sample:<br/><br />
DNA insert × µL<br/><br />
DNA vector × µL <br/><br />
T4 Ligation buffer (10×) × µL (for 1×)<br/><br />
T4 Ligase 1.0 µL <br/><br />
H2O × µL<br/><br />
tot. volume 10-15 µL<br/><br/><br />
<br />
The final concentration is preferably ~100 ng/µL. Smaller volumes are preferred and when DNA is at low concentration, try to evaporate water using a vacuum. Incubate at 16 °C for at least 3 hours or keep in an ice box in a floatie overnight. In the morning you find eppendorf tubes floating in water which had a temperature gradient overnight. For transformation use circa half of the ligation mix. <br/><br/><br />
<br />
<br />
<br />
<br />
<a name="P6"> <h1>Running a DNA gel</h1></a><br />
<br/><br />
<i>Materials:</i><br />
<ul><br />
<li>- Agarose </li><br />
<li>- TAE 1x </li><br />
<li>- SybrSafe DNA stain</li><br />
<li>- Loading Dye</li><br />
<li>- DNA ladder (Smartladder)</li><br />
<li>- DNA electrophoresis machine</li><br />
</ul><br />
<br/><br />
<i>Protocol:</i><br/><br />
<ol><br />
<li> Dissolve agarose (w/v 0.6% for separating long DNA pieces (>10 kbp), 1% for separating shorter pieces) in 1x TAE by microwaving</li><br />
<li> Close sides of electrophoresis tray (scotch tape works fine) and add comb</li><br />
<li> Let solution cool and add 5 µl Sybrsafe to an empty electrophoresis tray (small gels) 10-12 µl Sybrsafe for larger gels </li><br />
<li> Pour gel until a height of ~0.5 cm. Mix and remove bubbles with pipet tip (fast! It hardens quickly)</li><br />
<li> Put tray into electrophoresis casing and add TAE until a small layer above the gel can be seen. Remove comb</li><br />
<li>Add 1 µl loading dye to 5 µl sample, mix and load in the gel. Also add 5 µl smartladder for your reference</li><br />
<li>Run gel on 80 V (long run)- 110 V (short run, mostly for a ‘fast check’) for ~40-60 minutes, dependant of gel size, separation acquisition and voltage.</li><br />
</ol><br/><br />
For information about the smartladder, <a href=" https://secure.eurogentec.com/product/research-smartladder.html">Smartladder specifications</a>.<br />
<br/><br/><br />
<br />
<br />
<br />
<a name="P7"> <h1>PCR</h1></a><br />
<br/><br />
<i>Materials:</i><br/><br />
For PCR with minimal errors, pFX polymerase is used:<br />
<ul><br />
<li>Pfx polymerase (Invitrogen) </li><li><br />
10x Pfx Buffer (Invitrogen) </li><li><br />
enhancer (Invitrogen) </li><li><br />
50 mM MgSO4 (Invitrogen) </li><li><br />
10 mM dNTPs </li><br />
</ul><br />
<br/><br />
<br />
For PCR for checks of length, Taq polymerase is used, provided in Qiagen mastermix:<br/><br />
<ul><li>Mastermix </li></ul><br />
<br/><br />
Always needed:<br/><br />
<ul><br />
<li>primer solutions 5 mol/mL</li><li><br />
template DNA (plasmid at 50 pg – 1 ng/µL), or plate with colonies</li><li><br />
PCR machine</li><br />
</ul><br />
<br/><br/><br />
<br />
<br />
<i>Protocol:</i><br/><br />
First make sure that there is a PCR machine available for you. Take the solutions from the freezer and thaw them on ice.<br/><br/><br />
Preparation of reaction mixture:<br />
<ol><li>Gently vortex and briefly centrifuge all solutions after thawing</li><br />
<li>Keep solutions on ice</li><br />
<li>Add to a thin walled PCR tube, on ice the desired reaction mixture listed below.</li><br />
<li>for PCR on colonies: prick a sterile toothpick into a colony, dip it into a PCR tube and put it in 15 mL culture tube containing growth media to grow overnight for direct culturing positives.</li><br />
<li>Make sure you keep everything cool until it enters the preheated PCR machine</li></ol><br />
<br/><br />
<br />
<br />
<hr><br />
<br />
<div class=WordSection1><br />
<br />
<p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>For <span<br />
class=SpellE>Pfx</span> PCR the reaction mixture is:<o:p></o:p></span></p><br />
<br />
<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0<br />
style='margin-left:18.0pt;border-collapse:collapse;border:none;mso-border-alt:<br />
solid windowtext .5pt;mso-yfti-tbllook:480;mso-padding-alt:0cm 5.4pt 0cm 5.4pt;<br />
mso-border-insideh:.5pt solid windowtext;mso-border-insidev:.5pt solid windowtext'><br />
<tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'><br />
<td width=196 valign=top style='width:147.15pt;border:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><b>Component<o:p></o:p></b></p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border:solid windowtext 1.5pt;<br />
border-left:none;mso-border-left-alt:solid windowtext 1.5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><b>Sample<o:p></o:p></b></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:1'><br />
<td width=196 valign=top style='width:147.15pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext 1.5pt;mso-border-alt:<br />
solid windowtext 1.5pt;mso-border-bottom-alt:solid windowtext .5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><st1:metricconverter ProductID="10 mM" w:st="on">10 <span<br />
class=SpellE>mM</span></st1:metricconverter> <span class=SpellE>dNTPs</span></p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext 1.5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext 1.5pt;mso-border-bottom-alt:solid windowtext .5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'>1.5 <span<br />
class=SpellE>&#956;L</span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:2'><br />
<td width=196 valign=top style='width:147.15pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Enhancer</span> (<span class=SpellE>Invitrogen</span>)</p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;<br />
mso-border-right-alt:1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'>5.0 <span<br />
class=SpellE>&#956;L</span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:3'><br />
<td width=196 valign=top style='width:147.15pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal style='line-height:normal'>10x <span class=SpellE>Pfx</span><br />
Buffer (<span class=SpellE>Invitrogen</span>)</p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;<br />
mso-border-right-alt:1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'>5.0 <span<br />
class=SpellE>&#956;L</span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:4'><br />
<td width=196 valign=top style='width:147.15pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal style='line-height:normal'><st1:metricconverter<br />
ProductID="50 mM" w:st="on"><span lang=DE style='mso-ansi-language:DE'>50 <span<br />
class=SpellE>mM</span></span></st1:metricconverter><span lang=DE<br />
style='mso-ansi-language:DE'> MgSO<sub>4</sub> (<span class=SpellE>Invitrogen</span>)<br />
<o:p></o:p></span></p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;<br />
mso-border-right-alt:1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'>1.0 <span<br />
class=SpellE>&#956;L</span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:5'><br />
<td width=196 valign=top style='width:147.15pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>Primer 1 </p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;<br />
mso-border-right-alt:1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'>3.0 <span<br />
class=SpellE>&#956;L</span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:6'><br />
<td width=196 valign=top style='width:147.15pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>Primer 2 </p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;<br />
mso-border-right-alt:1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'>3.0 <span<br />
class=SpellE>&#956;L</span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:7'><br />
<td width=196 valign=top style='width:147.15pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal style='line-height:normal'><span class=SpellE>Pfx</span> <span<br />
class=SpellE>polymerase</span> (<span class=SpellE>Invitrogen</span>)</p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;<br />
mso-border-right-alt:1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'>0.6 <span<br />
class=SpellE>&#956;L</span> </p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:8'><br />
<td width=196 valign=top style='width:147.15pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>DNA <span class=SpellE>template</span></p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;<br />
mso-border-right-alt:1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'>1.0 <span<br />
class=SpellE>&#956;L</span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:9;mso-yfti-lastrow:yes'><br />
<td width=196 valign=top style='width:147.15pt;border:solid windowtext 1.5pt;<br />
border-top:none;mso-border-top-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>H<sub>2</sub>O </p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'>29.9 <span<br />
class=SpellE>&#956;L</span></p><br />
</td><br />
</tr><br />
</table><br />
<br/><br />
<p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>PCR program pFX : <br />
</span></p><br />
<br />
<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0 width=414<br />
style='width:310.55pt;margin-left:18.0pt;border-collapse:collapse;border:none;<br />
mso-border-alt:solid windowtext .5pt;mso-yfti-tbllook:480;mso-padding-alt:<br />
0cm 5.4pt 0cm 5.4pt;mso-border-insideh:.5pt solid windowtext;mso-border-insidev:<br />
.5pt solid windowtext'><br />
<tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'><br />
<td width=137 valign=top style='width:102.65pt;border:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><b>Step<o:p></o:p></b></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:solid windowtext 1.5pt;<br />
border-left:none;border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.0pt;<br />
mso-border-left-alt:solid windowtext 1.5pt;mso-border-alt:solid windowtext 1.5pt;<br />
mso-border-right-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE><b>Annealing</b></span><b><o:p></o:p></b></p><br />
<p class=MsoNormal><span class=SpellE><b>Temperature</b></span><b><o:p></o:p></b></p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:solid windowtext 1.5pt;<br />
border-left:none;border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.0pt;<br />
mso-border-left-alt:solid windowtext .5pt;mso-border-top-alt:1.5pt;<br />
mso-border-left-alt:.5pt;mso-border-bottom-alt:1.5pt;mso-border-right-alt:<br />
.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><b>Time, <o:p></o:p></b></p><br />
<p class=MsoNormal><b>min:sec<o:p></o:p></b></p><br />
</td><br />
<td width=91 valign=top style='width:68.0pt;border:solid windowtext 1.5pt;<br />
border-left:none;mso-border-left-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE><b>Number</b></span><b> of <span<br />
class=SpellE>cycles</span><o:p></o:p></b></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:1'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext 1.5pt;mso-border-alt:<br />
solid windowtext 1.5pt;mso-border-bottom-alt:solid windowtext .5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Initial</span> <span class=SpellE>denaturation</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext 1.5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:1.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:<br />
.5pt;mso-border-right-alt:.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>95 °C</p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext 1.5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-top-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>2:00</p><br />
</td><br />
<td width=91 rowspan=3 valign=top style='width:68.0pt;border-top:none;<br />
border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext 1.5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-top-alt:1.5pt;mso-border-left-alt:.5pt;mso-border-bottom-alt:.5pt;<br />
mso-border-right-alt:1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<p class=MsoNormal>1</p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:2'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Annealing</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span style='mso-spacerun:yes'> </span>X °C *</p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>1:00</p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:3'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Extension</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><st1:metricconverter ProductID="68&#65456;C" w:st="on">68°C</st1:metricconverter></p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>1:00-2:00 </p><br />
<p class=MsoNormal>(1 min/<span class=SpellE>kb</span>)</p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:4;page-break-inside:avoid'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Denaturation</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><st1:metricconverter ProductID="95 &#65456;C" w:st="on">95<br />
°C</st1:metricconverter></p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>1:00</p><br />
</td><br />
<td width=91 rowspan=3 valign=top style='width:68.0pt;border-top:none;<br />
border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-right-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<p class=MTDisplayEquation style='tab-stops:35.4pt'><span lang=EN-US<br />
style='font-size:11.0pt;mso-bidi-font-size:12.0pt;font-family:"Calibri","sans-serif"'>25<o:p></o:p></span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:5;page-break-inside:avoid'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Annealing</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span style='mso-spacerun:yes'> </span>X °C *</p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>1:00</p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:6;page-break-inside:avoid'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Extension</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><st1:metricconverter ProductID="68 &#65456;C" w:st="on">68<br />
°C</st1:metricconverter></p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>1:00-2:00 </p><br />
<p class=MsoNormal>(1 min/<span class=SpellE>kb</span>)</p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:7;mso-yfti-lastrow:yes;page-break-inside:avoid'><br />
<td width=137 valign=top style='width:102.65pt;border:solid windowtext 1.5pt;<br />
border-top:none;mso-border-top-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Final</span> <span class=SpellE>Extension</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:1.5pt;<br />
mso-border-right-alt:.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><st1:metricconverter ProductID="68 &#65456;C" w:st="on">68<br />
°C</st1:metricconverter></p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-bottom-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>10:00</p><br />
</td><br />
<td width=91 valign=top style='width:68.0pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>1</p><br />
</td><br />
</tr><br />
</table><br />
<br/><br />
<br />
<p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>For <span<br />
class=SpellE>Taq</span> PCR the reaction mixture is:<o:p></o:p></span></p><br />
<br />
<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0<br />
style='margin-left:18.0pt;border-collapse:collapse;border:none;mso-border-alt:<br />
solid windowtext .5pt;mso-yfti-tbllook:480;mso-padding-alt:0cm 5.4pt 0cm 5.4pt;<br />
mso-border-insideh:.5pt solid windowtext;mso-border-insidev:.5pt solid windowtext'><br />
<tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'><br />
<td width=196 valign=top style='width:147.15pt;border:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><b>Component<o:p></o:p></b></p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border:solid windowtext 1.5pt;<br />
border-left:none;mso-border-left-alt:solid windowtext 1.5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><b>Sample<o:p></o:p></b></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:1'><br />
<td width=196 valign=top style='width:147.15pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext 1.5pt;mso-border-alt:<br />
solid windowtext 1.5pt;mso-border-bottom-alt:solid windowtext .5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE><span lang=EN-US style='mso-ansi-language:<br />
EN-US'>Taq</span></span><span lang=EN-US style='mso-ansi-language:EN-US'> PCR<br />
Master Mix (<span class=SpellE>Qiagen</span><o:p></o:p></span></p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext 1.5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext 1.5pt;mso-border-bottom-alt:solid windowtext .5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'>12.5 <span<br />
class=SpellE>&#956;L</span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:2'><br />
<td width=196 valign=top style='width:147.15pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>Primer 1</p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;<br />
mso-border-right-alt:1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'><span lang=EN-US<br />
style='mso-ansi-language:EN-US'>1.5 </span>&#956;<span lang=EN-US<br />
style='mso-ansi-language:EN-US'>L<o:p></o:p></span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:3'><br />
<td width=196 valign=top style='width:147.15pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>Primer 2<o:p></o:p></span></p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;<br />
mso-border-right-alt:1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'><span lang=EN-US<br />
style='mso-ansi-language:EN-US'>1.5 </span>&#956;<span lang=EN-US<br />
style='mso-ansi-language:EN-US'>L<o:p></o:p></span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:4'><br />
<td width=196 valign=top style='width:147.15pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>Template<br />
DNA (or pick a colony)<o:p></o:p></span></p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;<br />
mso-border-right-alt:1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'><span lang=EN-US<br />
style='mso-ansi-language:EN-US'>1 <span class=SpellE>&#956;L</span><o:p></o:p></span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:5;mso-yfti-lastrow:yes'><br />
<td width=196 valign=top style='width:147.15pt;border:solid windowtext 1.5pt;<br />
border-top:none;mso-border-top-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>H<sub>2</sub>O<br />
<o:p></o:p></span></p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'><span lang=EN-US<br />
style='mso-ansi-language:EN-US'>8.5 </span>&#956;<span lang=EN-US<br />
style='mso-ansi-language:EN-US'>L<o:p></o:p></span></p><br />
</td><br />
</tr><br />
</table><br />
<br />
<br/><br />
<br />
<p class=MsoNormal>PCR program <span class=SpellE>Taq</span> <span<br />
class=SpellE>polymerase</span><span style='mso-bidi-font-weight:bold'>:</span></p><br />
<br />
<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0 width=414<br />
style='width:310.55pt;margin-left:18.0pt;border-collapse:collapse;border:none;<br />
mso-border-alt:solid windowtext .5pt;mso-yfti-tbllook:480;mso-padding-alt:<br />
0cm 5.4pt 0cm 5.4pt;mso-border-insideh:.5pt solid windowtext;mso-border-insidev:<br />
.5pt solid windowtext'><br />
<tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'><br />
<td width=137 valign=top style='width:102.65pt;border:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><b>Step<o:p></o:p></b></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:solid windowtext 1.5pt;<br />
border-left:none;border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.0pt;<br />
mso-border-left-alt:solid windowtext 1.5pt;mso-border-alt:solid windowtext 1.5pt;<br />
mso-border-right-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE><b>Annealing</b></span><b><o:p></o:p></b></p><br />
<p class=MsoNormal><span class=SpellE><b>Temperature</b></span><b><o:p></o:p></b></p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:solid windowtext 1.5pt;<br />
border-left:none;border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.0pt;<br />
mso-border-left-alt:solid windowtext .5pt;mso-border-top-alt:1.5pt;<br />
mso-border-left-alt:.5pt;mso-border-bottom-alt:1.5pt;mso-border-right-alt:<br />
.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><b>Time, <o:p></o:p></b></p><br />
<p class=MsoNormal><b>min:sec<o:p></o:p></b></p><br />
</td><br />
<td width=91 valign=top style='width:68.0pt;border:solid windowtext 1.5pt;<br />
border-left:none;mso-border-left-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE><b>Number</b></span><b> of <span<br />
class=SpellE>cycles</span><o:p></o:p></b></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:1'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext 1.5pt;mso-border-alt:<br />
solid windowtext 1.5pt;mso-border-bottom-alt:solid windowtext .5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Initial</span> <span class=SpellE>denaturation</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext 1.5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:1.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:<br />
.5pt;mso-border-right-alt:.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>94 °C</p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext 1.5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-top-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>10:00</p><br />
</td><br />
<td width=91 rowspan=3 valign=top style='width:68.0pt;border-top:none;<br />
border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext 1.5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-top-alt:1.5pt;mso-border-left-alt:.5pt;mso-border-bottom-alt:.5pt;<br />
mso-border-right-alt:1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<p class=MsoNormal>1</p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:2'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Annealing</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span style='mso-spacerun:yes'> </span>X °C *</p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>0:45</p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:3'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Extension</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>72°C</p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>1:00-2:00 </p><br />
<p class=MsoNormal>(1 min/<span class=SpellE>kb</span>)</p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:4;page-break-inside:avoid'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Denaturation</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><st1:metricconverter ProductID="95 &#65456;C" w:st="on">95<br />
°C</st1:metricconverter></p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>1:00</p><br />
</td><br />
<td width=91 rowspan=3 valign=top style='width:68.0pt;border-top:none;<br />
border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-right-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<p class=MTDisplayEquation style='tab-stops:35.4pt'><span lang=EN-US<br />
style='font-size:11.0pt;mso-bidi-font-size:12.0pt;font-family:"Calibri","sans-serif"'>25<o:p></o:p></span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:5;page-break-inside:avoid'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Annealing</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span style='mso-spacerun:yes'> </span>X °C *</p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>1:00</p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:6;page-break-inside:avoid'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Extension</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>72 °C</p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>1:00-2:00 </p><br />
<p class=MsoNormal>(1 min/<span class=SpellE>kb</span>)</p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:7;mso-yfti-lastrow:yes;page-break-inside:avoid'><br />
<td width=137 valign=top style='width:102.65pt;border:solid windowtext 1.5pt;<br />
border-top:none;mso-border-top-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Final</span> <span class=SpellE>Extension</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:1.5pt;<br />
mso-border-right-alt:.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>72 °C</p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-bottom-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>10:00</p><br />
</td><br />
<td width=91 valign=top style='width:68.0pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>1</p><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<br/><br />
<br />
<p>*Annealing temperature is very dependent on primer. Optimal temperature: 3x G/C + 2x A/T </p><br />
<br />
<br />
<br/><br />
<br />
<a name="P8"> <h1>Gel Extraction</h1></a><br />
<br/><br />
This protocol is based on QIAGEN® Gel Extraction Handbook.<br />
<br/><br/><br />
<i>Materials:</i><br />
<br/><br />
<ul><li>QIAquick columns</li><li><br />
buffer QG</li><li><br />
buffer PE</li><li><br />
isopropanol</li><li><br />
milliQ</li><li><br />
microcentrifuge</li><li><br />
heat block at 50 °C</li><br />
</ul><br />
<br/><br />
<i>Protocol:</i><br />
<ol><br />
<li>Excise the DNA fragment from the agarose gel with a clean, sharp scalpel. Minimize the size of the gel slice by removing extra agarose. <br />
</li><br />
<li><br />
Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg ~ 100 µL). For >2% agarose gels, add 6 volumes of Buffer QG. The maximum amount of gel slice per QIAquick column is 400 mg; for gel slices >400 mg use more than one QIAquick column. <br />
</li><br />
<li><br />
Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2–3 min during the incubation. <br/><br />
IMPORTANT: Solubilize agarose completely. For >2% gels, increase incubation time. <br />
</li><br />
<li><br />
After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 µL of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow. The adsorption of DNA to the QIAquick membrane is efficient only at pH <7.5. Buffer QG contains a pH indicator which is yellow at pH <7.5 and orange or violet at higher pH, allowing easy determination of the optimal pH for DNA binding. <br />
</li><br />
<li><br />
Add 1 gel volume of isopropanol to the sample and mix. This step increases the yield of DNA fragments <500 bp and >4 kb. For DNA fragments between 500 bp and 4 kb, addition of isopropanol has no effect on yield. Do not centrifuge the sample at this stage. <br />
</li><br />
<li><br />
Place a QIAquick spin column in a provided 2 ml collection tube. <br />
</li><br />
<li><br />
To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 min. <br />
The maximum volume of the column reservoir is 800 µL. For sample volumes of more than 800 µL, simply load and spin again. <br />
</li><br />
<li><br />
Discard flow-through and place QIAquick column back in the same collection tube. <br />
</li><br />
<li><br />
To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min. <br />
</li><br />
<li><br />
Discard the flow-through and centrifuge the QIAquick column for an additional 1 min at 10,000 x g (~13,000 rpm). <br />
</li><br />
<li><br />
IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation. <br />
</li><br />
<li><br />
Place QIAquick column into a clean 1.5 ml microcentrifuge tube. <br />
</li><br />
<li><br />
To elute DNA, add 50 µL of Buffer EB (10 mM Tris•Cl, pH 8.5) or H2O to the center of the QIAquick membrane and centrifuge the column for 1 min at maximum speed. Alternatively, for increased DNA concentration, add 30 µl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min. <br/><br />
Important: Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA. The average eluate volume is 48 µL from 50 µL elution buffer volume, and 28 µL from 30 µL. Elution efficiency is dependent on pH. The maximum elution efficiency is achieved between pH 7.0 and 8.5. When using water, make sure that the pH value is within this range, and store DNA at –20°C as DNA may degrade in the absence of a buffering agent. The purified DNA can also be eluted in TE (10 mM Tris•Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.<br />
</li><br />
</ol><br />
<br />
<br/><br/><br />
<br />
<br />
<a name="P11"> <h1>Immunofluorescence Staining</h1></a><br />
<br/><br />
<div class=WordSection1><br />
<br />
<p class=MsoNoSpacing><span lang=EN-US>This protocol is for immunofluorescence staining<br />
of yeast to prepare it for Flow <span class=SpellE>Cytometry</span> or fluorescence<br />
microscopy, and staining the target of the conjugated antibody of choice. The<br />
TU Delft <span class=SpellE>iGEM</span> 2012 team used a <a<br />
href="http://www.cellsignal.com/products/3916.html"><span class=SpellE>flagtag</span><br />
antibody.</a> <span class=GramE>The</span> protocol of the supplier was<br />
improved for yeast staining. <b style='mso-bidi-font-weight:normal'>IMPORTANT:</b>&nbsp;Please<br />
refer to the&nbsp;<b style='mso-bidi-font-weight:normal'>APPLICATIONS</b><span<br />
class=GramE>&nbsp; section</span> on the front page of the datasheet of the<br />
antibody product to determine if this product is validated and approved for use<br />
on cultured cell lines (IF-IC).</span></p><br />
<br />
<p class=MsoNoSpacing><span lang=EN-US style='mso-fareast-language:NL'><o:p>&nbsp;</o:p></span></p><br />
<br />
<p class=MsoNoSpacing><span lang=EN-US style='mso-bidi-font-family:Calibri;<br />
mso-bidi-theme-font:minor-latin;mso-fareast-language:NL'>Materials:<o:p></o:p></span></p><br />
<br />
<p class=MsoNoSpacing><span lang=EN-US style='mso-bidi-font-family:Calibri;<br />
mso-bidi-theme-font:minor-latin;mso-fareast-language:NL'><o:p>&nbsp;</o:p></span></p><br />
<br />
<p class=MsoNoSpacing style='margin-left:36.0pt;text-indent:-18.0pt;mso-list:<br />
l3 level1 lfo13'><![if !supportLists]><span lang=EN-US style='font-family:Symbol;<br />
mso-fareast-font-family:Symbol;mso-bidi-font-family:Symbol;mso-fareast-language:<br />
NL'><span style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span lang=EN-US style='mso-fareast-language:<br />
NL'><a href="http://openwetware.org/wiki/PBS">Phosphate Buffered Saline (PBS)</a>,<br />
adjust pH to 8.0.<o:p></o:p></span></p><br />
<br />
<p class=MsoNoSpacing style='margin-left:36.0pt;text-indent:-18.0pt;mso-list:<br />
l3 level1 lfo13'><![if !supportLists]><span lang=EN-US style='font-family:Symbol;<br />
mso-fareast-font-family:Symbol;mso-bidi-font-family:Symbol;mso-fareast-language:<br />
NL'><span style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span lang=EN-US style='mso-fareast-language:<br />
NL'>Formaldehyde, 16%, <b style='mso-bidi-font-weight:normal'>methanol free</b><o:p></o:p></span></p><br />
<br />
<p class=MsoNoSpacing style='margin-left:36.0pt;text-indent:-18.0pt;mso-list:<br />
l3 level1 lfo13'><![if !supportLists]><span lang=EN-US style='font-family:Symbol;<br />
mso-fareast-font-family:Symbol;mso-bidi-font-family:Symbol;mso-fareast-language:<br />
NL'><span style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span lang=EN-US style='mso-fareast-language:<br />
NL'>Blocking Buffer:&nbsp;(1X PBS / 1 <span class=SpellE>wt</span>% BSA / 0.3%<br />
(v/v) Triton X-100)<o:p></o:p></span></p><br />
<br />
<p class=MsoNoSpacing style='margin-left:36.0pt;text-indent:-18.0pt;mso-list:<br />
l3 level1 lfo13'><![if !supportLists]><span lang=EN-US style='font-family:Symbol;<br />
mso-fareast-font-family:Symbol;mso-bidi-font-family:Symbol;mso-fareast-language:<br />
NL'><span style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span lang=EN-US style='mso-fareast-language:<br />
NL'>Antibody Dilution Buffer&nbsp;(1X PBS / 1 <span class=SpellE>wt</span>% BSA<br />
/ 0.3% (v/v) Triton X-100)<o:p></o:p></span></p><br />
<br />
<p class=MsoNoSpacing style='margin-left:36.0pt;text-indent:-18.0pt;mso-list:<br />
l3 level1 lfo13'><![if !supportLists]><span lang=EN-US style='font-family:Symbol;<br />
mso-fareast-font-family:Symbol;mso-bidi-font-family:Symbol;mso-fareast-language:<br />
NL'><span style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span lang=EN-US style='mso-fareast-language:<br />
NL'>Primary conjugated antibody.<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:<br />
22.2pt;background:white'><b><span style='mso-fareast-font-family:"Times New Roman";<br />
mso-bidi-font-family:Calibri;mso-bidi-theme-font:minor-latin;color:#2B6EAE;<br />
mso-ansi-language:NL;mso-fareast-language:NL'><o:p>&nbsp;</o:p></span></b></p><br />
<br />
<p class=MsoNoSpacing><span lang=EN-US style='mso-fareast-language:NL'>Protocol:<o:p></o:p></span></p><br />
<br />
<p class=MsoNoSpacing><span lang=EN-US style='mso-fareast-language:NL'><o:p>&nbsp;</o:p></span></p><br />
<br />
<p class=MsoNoSpacing><span lang=EN-US style='mso-fareast-language:NL'>The<br />
desired culture of cell is taken for staining and the liquid is aspirated by<br />
spinning it down in appropriate tubing <span class=GramE>( e.g</span>. standard<br />
<span class=SpellE>Eppendorf</span> tubes )<span style='mso-spacerun:yes'> <br />
</span>and decanting or pipetting the supernatant.<span<br />
style='mso-spacerun:yes'> </span>Afterwards the container is inspected for a<br />
cell pellet of acceptable size, meaning it must be easy to spot in the<br />
container. After this the fixing of the cells can start:<o:p></o:p></span></p><br />
<br />
<p class=MsoNoSpacing><b><span lang=EN-US style='color:#2B6EAE;mso-fareast-language:<br />
NL'><o:p>&nbsp;</o:p></span></b></p><br />
<br />
<p class=MsoNoSpacing style='margin-left:36.0pt;text-indent:-18.0pt;mso-list:<br />
l9 level1 lfo14'><![if !supportLists]><span lang=EN-US style='mso-bidi-font-family:<br />
Calibri;mso-bidi-theme-font:minor-latin;mso-fareast-language:NL'><span<br />
style='mso-list:Ignore'>1.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span lang=EN-US style='mso-fareast-language:<br />
NL'>Cover cells to a depth of 10–15 mm with 4% formaldehyde in PBS and <span<br />
class=SpellE>resuspend</span> cells.<br/><br />
<b>NOTE:</b>&nbsp;Formaldehyde is toxic, use only in fume hood.<o:p></o:p></span></p><br />
<br />
<p class=MsoNoSpacing style='margin-left:36.0pt;text-indent:-18.0pt;mso-list:<br />
l9 level1 lfo14'><![if !supportLists]><span lang=EN-US style='mso-bidi-font-family:<br />
Calibri;mso-bidi-theme-font:minor-latin;mso-fareast-language:NL'><span<br />
style='mso-list:Ignore'>2.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span lang=EN-US style='mso-fareast-language:<br />
NL'>Allow cells to fix for 15 minutes at room temperature.<o:p></o:p></span></p><br />
<br />
<p class=MsoNoSpacing style='margin-left:36.0pt;text-indent:-18.0pt;mso-list:<br />
l9 level1 lfo14'><![if !supportLists]><span lang=EN-US style='mso-bidi-font-family:<br />
Calibri;mso-bidi-theme-font:minor-latin;mso-fareast-language:NL'><span<br />
style='mso-list:Ignore'>3.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span lang=EN-US style='mso-fareast-language:<br />
NL'>Aspirate fixative and rinse three times in PBS for 5 minutes each before<br />
proceeding to the staining section. <span class=GramE>( Use</span> the same<br />
aspirating techniques as above: Spin, Aspirate, add, <span class=SpellE>resuspend</span>.<br />
)<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;<br />
margin-left:48.0pt;margin-bottom:.0001pt;line-height:22.2pt;background:white'><span<br />
lang=EN-US style='mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:<br />
Calibri;mso-bidi-theme-font:minor-latin;color:black;mso-fareast-language:NL'><o:p>&nbsp;</o:p></span></p><br />
<br />
<p class=MsoNoSpacing><span lang=EN-US style='mso-fareast-language:NL'>All subsequent<br />
incubations should be carried out at room temperature unless otherwise noted in<br />
a humid light-tight box or covered dish/plate to prevent drying and <span<br />
class=SpellE>fluorochrome</span> fading.<o:p></o:p></span></p><br />
<br />
<p><ol><br />
<br />
<li> Block specimen in Blocking Buffer for 60 minutes.</li><br />
<li> While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer. ( A Cell Signaling technology antibody was used with this protocol )</li><br />
<li> Aspirate blocking solution, apply diluted primary antibody.</li><br />
<li> Incubate overnight at 4°C.</li><br />
<li> Rinse three times in PBS for 5 minutes each.<br />
NOTE: Because one is using primary antibodies directly conjugated with ( Alexa Fluor® ) fluorochromes, secondary antibodies are not needed.</li><br />
<li> For best results, examine specimens immediately using appropriate excitation wavelength. For long-term storage, store slides flat at 4°C protected from light.</li><br />
</ol></p><br />
<br />
<br />
</div><br />
<br />
<br />
<br/><br />
<a name="P12"><h1>DNA Staining</h1></a><br/><br/><br />
<i>Materials</i><br/><br />
<ul><li>Your favorite yeast strain</li><br />
<li>Vybrant DyeCycle Orange from invitrogen</li><br />
<li>PBS</li><br />
<li>37°C heat block</li><br />
</ul><br/><br/><br />
<br />
<i>Protocol</i><br/><br />
<ol><li>Centrifuge cells on a table top centrifuge for 10 s on 13.000 rpm </li><br />
<li>Take of supernatant and add PBS</li><br />
<li>Add 2 µL/mL Vybrant DyeCycle Orange and vortex until homogenuous solution</li><br />
<li>Keep for 30 minutes on 37°C and the cells are stained.</li><br/><br/><br />
</ol><br />
<br />
<a name="P13"><h1>Flow cytometry to look at DNA content</h1></a><br/><br />
<i>Materials</i><br/><br />
<ul><li>Yeast culture</li><br />
<li>Vybrant® DyeCycleTM Orange stain</li><br />
<li>Ligand</li><br />
<li>Flow cytometry instrument</li><br/><br />
</ul><br />
<i>Protocol</i><br/><br />
<ol><br />
<li>Grow yeast cells at 30C overnight and re-inocculate in 10 mL medium the next morning</li><br />
<li>If cells are in exponential growth phase add the ligand (also keep a control without ligand)</li><br />
<li>After incubation stain the cells according to protocol</li><br />
<li>Transfer the stained cells in a flow cytometry tube</li><br />
<li>Analyze the samples on a flow cytometer using 488 nm excitation or 532 nm excitation and orange emission</li><br />
<li>After each measurement flush the tube with PBS buffer</li><br />
</ol><br />
<br/><br />
<br/><br />
<a name="P14"><h1>Flow cytometry to look at EGFP expression</h1></a><br/><br />
<br />
<br />
<i>Materials</i><br/><br />
<ul><br />
<li>Yeast culture</li><br />
<li>Ligand</li><br />
<li>Flow cytometry instrument</li><br />
</ul><br />
<br/><br />
<i>Protocol</i><br/><br />
<ol><li>Grow yeast cells at 30C overnight and re-inocculate in 10 mL medium the next morning</li><br />
<li>If cells are in exponential growth phase add the ligand (also keep a control without ligand)</li><br />
<li>Transfer the cells in a flow cytometry tube</li><br />
<li>Analyze the samples on a flow cytometer using 488 nm excitation and 509 nm emission</li><br />
<li>After each measurement flush the tube with PBS buffer</li><br />
</ol><br />
<br/><br />
<br/><br />
<a name="P15"><h1>Fluorimeter experiment</h1></a><br/><br/><br />
<i>Materials</i><br/><br />
<ul><br />
<li>Yeast culture</li><br />
<li>Ligand</li><br />
<li>Fluorimeter plate reader</li><br />
<li>96 well plate</li><br/><br />
</ul><br />
<i>Protocol</i><br />
<br />
<br/><br />
<ol><li>Grow yeast cells at 30C overnight and re-inocculate the next morning</li><br />
<li>Measure after approx. 3h hours the OD600. Dilute the cells with medium until an OD of approx. 0.1</li><br />
<li>Transfer the cells to the 96 well plate. Don't forget to have a well with only medium.</li><br />
<li>Add the ligand.</li><br />
<li>Analyze the samples in the fluorimeter that is kept at 30C. Use filters with 488 nm excitation and 509 nm emission. At the same time measure the OD600.</li><br />
<li>Calculate the fluoresce per biomass by dewing the fluorescence by the OD600 values</li><br />
</ol><br><br />
<br />
<a name="P17"><h1>Growth rate experiment</h1></a><br/><br/><br />
<i>Materials</i><br/><br />
<ul><br />
<li>Yeast cultures (wt with and without alpha feromones and far1KO with and without alpha feromones)</li><br />
<li>Alpha feromones</li><br />
<li>Spectrophotometer</li><br />
<li>30C incubator</li><br/><br />
</ul><br />
<i>Protocol</i><br />
<br />
<br/><br />
<ol><li>Grow yeast cells at 30C overnight and re-inocculate 20 ml of medium with 500µl of grown cells the next morning</li><br />
<li>Measure OD600 every hour. </li><br />
<li>Add alpha feromones after 3hr</li><br />
<li>Measure OD600every hour, dilute if needed.</li><br />
</ol><br><br />
<br />
<a name="P18"><h1>Smell activity tests with petri dishes</h1></a><br/><br/><br />
<i>Materials</i><br/><br />
<br />
<ul><br />
<br />
<li>4 x Two yeast cultures with each having six yeast-strains</li><br />
<br />
<li>Ligands </li><br />
<br />
Water 2 x<br />
<br />
Alpha pheromone 1000x diluted 2 x<br />
<br />
Isoamyl acetate 99%<br />
<br />
Methyl phenylacete 99%<br />
<br />
Niacin 0.336 g/100 ml<br />
<br />
Methyl nicotinate 10 mM<br />
<br />
<li>Typhoon fluorescence scanner</li><br />
<br />
<li>8 Resealable (hard) plastic boxes of 10x10x10 cm.</li><br/><br />
<br />
</ul><br />
<br />
<br/><br />
<br />
<br/><br />
<br />
<i>Protocol</i><br />
<br />
<br/><br />
<br />
<ol><li>Grow yeast cells at 30C for two days.</li><br />
<br />
<li>Analyze the samples in the Typhoon fluorescence scanner before starting experiment.</li><br />
<br />
<li>Pore approx. 25 ml of every ligand (except alpha pheromone) in the box after cleaning it with alcohol / autoclaving if possible. </li><br />
<br />
<li>Place two sided tape on the inwards part of the boxlid. Gently but firmly push the petri dish on it. Remove the lid of the dish and put the dish upside down in the box. For alpha pheromone; add solution directly on all yeast strains.</li><br />
<br />
<li>After 3.5 hours remove the dish from the lid.</li><br />
<br />
<li>Analyze the samples in the Typhoon fluorescence scanner. Use filters with 488 nm excitation and 509 nm emission. Set PMT to 300. Orientation of dish to Platen.</li><br />
<br />
</ol><br><br><br />
<br />
<br />
<a name="P16"><h1>Tips&Tricks for working with yeast</h1></a><br />
<br />
<p>Since this is the first time that TU Delfts iGEM team is working with yeast, we faced a lot of small yeast-related 'challenges'. With this page we want to inform you about the basics and the pitfalls of working with yeast. </p><br />
<h3>Basics</h3><br />
<p>Yeast, <i>Saccharomyces cerevisiae</i>, is a simple, unicellular eukaryotic organism. This organism has been used for fermentation and baking for over 4000 years and it is probably the oldest domesticated micro-organism in the human history. An important one too, can you imagine a life without bread and beer? (Even if you can imagine it, it would most definitely be less fun!) <br />
Nowadays the whole genomic sequence of <i>Saccharomyces cerevisiae</i> is known and a lot of genomic tools are available.</p><br />
<h3>Auxotrophy</h3><br />
<p> The main advantage from an engineering perspective is that yeast has Auxotrophic markers. In specific strains genes are knocked out which synthesize essential enzymes in the amino acid synthesis routes. By complementing these deficiencies by adding the necessary gene on your DNA this provides a nice selection procedure. <br />
<h3> PRS huttle vectors</h3> <br />
<p>The name pRS415 gives an indication on the presence of a CEN/ARS replication origin. 0 means yeast<br />
integrative plasmid, 1 means that it also can be used to maintain the plasmid in circular<br />
form.<br />
pRS415 Gives an indication of the auxotrophic marker used.<br />
pRS415 Version number… not really different.<br/><br />
<h3>Chromosomal integration</h3><br />
<p>We encountered a lot of problems with plasmids. Because we wanted our constructs to be universal<br />
(with the idea to make it suitable for ‘fast checking’) we tried maintaining a plasmid. As it turned<br />
out, yeast cells are not eager to maintain a plasmid and with our construct we suspect homologous<br />
recombination occurred. After transformation, a PCR on the transformed plasmid, obtained by<br />
isolation, showed two bands instead of the suspected single band, one being ! Integration of the<br />
plasmid is therefore advised! Checking of this can be quite gruesome optimizing the necessary PCR<br />
reactions on your transformed yeast colonies. Chromosomal isolation can therefore improve the<br />
steps. </p><br/><br />
<h3>Knock-out strains</h3> <br />
<p>European iGEM teams have the advantage to have <a href="http://web.uni-<br />
frankfurt.de/fb15/mikro/euroscarf">Euroscarf</a> available to order strains with knocked out ORFs. The typical<br />
nomenclature is also explained here: <a href="http://web.uni-frankfurt.de/fb15/mikro/euroscarf/<br />
stra_des.html">Euroscarf explanation</a>.</p><br/><br />
<br />
<br />
<br />
<br />
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<a href='https://2012.igem.org/Main_Page' target="_blank"><div id='logo_igem2'></div><a/></body></html></div>MarkWeijershttp://2012.igem.org/Team:TU-Delft/protocolsTeam:TU-Delft/protocols2012-10-27T03:05:15Z<p>MarkWeijers: </p>
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<br />
<h1>Protocols</h1> <br />
<br />
<a href="#P10"> Media</a><br/><br />
<a href="#P9"> Yeast Transformation</a><br/><br />
<a href="#P1"> Transforming One Shot® Mach1™ competent cells </a><br/><br />
<a href="#P2"> Mini-prep plasmid isolation for E. coli and S. cerevisae </a><br/><br />
<a href="#P3"> Freezing cell stocks </a><br/><br />
<a href="#P4"> Restriction enzyme digestion </a><br/><br />
<a href="#P5"> Ligation </a><br/><br />
<a href="#P6"> Running DNA Gel </a><br/><br />
<a href="#P7"> PCR </a><br/><br />
<a href="#P8">Gel Extraction</a><br/><br />
<a href="#P11">Immunofluorescence Staining</a><br/><br />
<a href="#P12">DNA Staining</a><br/><br />
<a href="#P13">Flow cytometry to look at DNA content</a><br/><br />
<a href="#P14">Flow cytometry to look at EGFP expression</a><br/><br />
<a href="#P15">Fluorimeter experiment</a><br/><br />
<a href="#P17">Growth curve experiment</a><br/><br />
<a href="#P18">Smell activity tests with petri dishes</a><br/><br />
<a href="#P16">Tips&Tricks for working with yeast</a><br/><br/><br />
<br />
<a name="P10"><h1>Media</h1></a><br />
<br/><br />
<i>DO (agar)</i><br />
<br/>For BY4741 yeast strain add to mineral media:<br/>HIS: 125 mg/liter<br/>LEU 500 mg/liter<br/>MET 100 mg/liter<br/>URA 150 mg/liter<br/>except for one which you want to select on with you auxotrophic marker.<br/><br/>Also add:<br/>2% Sucrose (w/v)<br/>2% Agarose<br/><br/>and autoclave<br/><br/><br />
<i>YPD (agar)</i><br />
<br/>http://openwetware.org/wiki/YPD <br/><br/><br />
<i>LB (agar) </i><br/><br />
<br/>http://openwetware.org/wiki/LB <br/><br/><br />
<br />
<a name="P9"><h1>Yeast Transformation</h1></a><br />
<br/><br />
<i>Materials</i><br/><br />
<ul><li>Yeast culture</li><br />
<li>single stranded carrier DNA (200 mg Salmon sperm DNA inTE)</li><br />
<li>Lithium Acetate (1.0 M)</li><br />
<li>Polyethylene Glycol 3350 (50% w/v)</li><br />
<li>DO media plates </li><br />
<li>Dropout (DO) media, lacking an amino acid of which an auxotrophic marker is added</li><br />
<li>Water bath 42 °C</li><br />
<li>Water bath 30 °C</li><br/><br/><br />
</ul><br />
<br />
<i>Protocol</i><br/><br />
<br />
<br />
<ol><li> Grow yeast culture overnight in 10 mL YPD on 30 °C</li><br />
<li> Put 0.5 mL – 1 mL into new flask with 20 mL YPD (for 2-4 transformations) and grow 4-5 hours until an OD of 0.8 is measured. The cells are now in exponential phase.</li><br />
<li> Spin the cells for 5 minutes on 3000 G and discard supernatant</li><br />
<li> Wash cells with water and spin down for 5 minutes on 3000 G</li><br />
<li> During centrifuging, make a transformation mix (TM): Per reaction add <br/><br />
<ul><li>240 µL 50% PEG</li><br />
<li>36 µL 1M Lithium Acetate solution</li><br />
<li>25 µL ssDNA, boiled for 5 minutes and then cooled on ice</li><br />
<li>49 µL water</li><br />
</ul><br/><br />
</li><br />
<li> Discard supernatant and resuspend in 0.2 mL 0.1 M Lithium Acetate solution</li><br />
<li> Do a quick centrifuge (10 s 13.000 rpm on a table top centrifuge), discard supernatant and add 80 µL 0.1 M Lithium Acetate solution. Vortex and do <u>not</u> pipette up and down</li><br />
<li>You should have ~100 µL of which 50 µL should be put in a fresh tube. This is you negative control</li><br />
<li>spin cells down again and add the mastermix</li><br />
<li>Add 1 µL target (100 ng – 1000 ng) DNA to the positive tube</li><br />
<li>Put in a 30 °C water bath for 30 minutes</li><br />
<li>Put it in a 42 °C water bath for 30 minutes</li><br />
<li>Do a quick centrifuge (10 s 13.000 rpm on a table top), remove supernatant. Add 0.2 mL water, pipet up and down and plate 150 µL and 50 µL on DO selective plates </li><br />
</ol><br/><br/><br />
<br />
<br />
<a name="P1"> <h1>Transforming One Shot® Mach1™ competent cells</h1></a><br/><br />
<br />
<br/><br />
<i>Materials:</i><br />
<br/><br />
<ul><br />
<li>- Competent cells</li><br />
<li> - SOC medium (warmed to room temperature)</li><br />
<li> - Plasmid DNA or DNA ligation mix</li><br />
<li> - LB agar plates containing 15-100 ?g/mL antibiotic of choice, pre-warmed to 37 °C </li><br />
<li> - water bath at 42 °C </li><br />
<li> - shaking incubator at 37 °C. </li><br />
</ul><br />
<br/><br />
<i>Protocol:</i><br />
<br/><br />
<ol><li>Add 50-100 ng DNA into a 20 ?L competent E.coli, and mix gently. Do not mix by pipetting up and down!</li><br />
<li>Incubate tube vial on ice for 30 minutes</li><br />
<li>Heat-shocks the cells for 30 seconds at 42 °C without shaking</li><br />
<li>Immediately transfer the tubes back to ice for 2 minutes</li><br />
<li>Add 250 ?L of room temperature LB medium</li><br />
<li>Cap tube tightly and shake tube horizontally (225 rpm) at 37 °C for 1 hour</li><br />
<li>Plate from each tube 100 ?L on an agar plate containing antibiotic. Spin tube, discard supernatant to leave no more than 100 ?L, vortex and plate on an agar plate</li><br />
<li>Incubate plates overnight at 37 °C</li><br />
</ol><br />
<br/><br />
<br />
<br />
<br />
<a name="P2"> <h1> Qiagen Mini-prep plasmid isolation for E. coli and S. cerevisae</h1></a><br />
<br/><br />
This protocol is based on QIAGEN® Plasmid Purification Handbook.<br/><br />
<i>Materials:</i><br/><br />
<ul><br />
<li>- bacterial or yeast culture</li><br />
<li>- Qiagen colums</li><br />
<li>- buffer P1 (100 mg/mL RNAse A, 50 mM Tris/HCl, 10 mM EDTA, pH 8.0) </li><br />
<li>- buffer P2 (200 mM NaOH, 1% SDS) </li><br />
<li>- buffer P3 (3 M KAc, pH 5.5) </li><br />
<li>- buffer PE </li><br />
<li>- milliQ pH 8.0</li><br />
<li>- centrifuge</li><br />
<li>- nanodrop</li><br />
<li>- for yeast plasmid isolation: zymolyase 5 U/µl</li></ul><br/><br />
<i> Protocol:</i><br/><br />
<ol><br />
<li>Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 2–5 mL LB medium containing the appropriate selective antibiotic or selective medium. Incubate for approximately 8 h at 37°C (bacteria) or 12 h at 30°C with vigorous shaking (approx. 300 rpm)</li><br />
<li>Harvest the 5 mL bacterial cells by centrifugation at 13,000 rpm for 1 min at 20°C (microcentrifuge tube). If you wish to stop the protocol and continue later, freeze the cell pellets at –20°C</li><br />
<li>For bacteria: Resuspend pelleted bacterial cells in 250 µL Buffer P1. Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet. <br/> For yeast: Resuspend cells in 250 µL Buffer P1 with 3 µL zymolyase and incubate 1 h at 37 °C</li><br />
<li>Add 250 µL Buffer P2 and mix thoroughly by inverting the tube 4–6 times. Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. 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><br />
<li>Add 350 µL Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer N3. Large culture volumes (e.g. =5 mL) may require inverting up to 10 times. The solution should become cloudy. </li><br />
<li>Incubate at -20 °C for 15 minutes. </li><br />
<li>Centrifuge for 10 min at 13,000 rpm in a table-top microcentrifuge. A compact white pellet will form. </li><br />
<li>Apply the supernatants from step 7 to the QIAprep spin column by decanting or pipetting. </li><br />
<li>Centrifuge for 30–60 seconds. Discard the flow-through. </li><br />
<li>Wash QIAprep spin column by adding 0.75 mL Buffer PE and centrifuging for 30–60 seconds. </li><br />
<li>Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer. <br />
<b>Important:</b> Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions. </li><br />
<li>Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 30 µL Buffer EB (10 mM Tris•Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 minute in 50 C stove and centrifuge for 1 minute to obtain DNA. </li><br />
<li>Measure DNA concentration on the Nanodrop</li><br />
</ol><br/><br />
<br />
<br />
<br />
<br />
<a name="P3"> <h1>Freezing cell stocks</h1></a><br />
<br/><br />
<i>Materials:</i> <br />
<ul><br />
<li>- bacterial culture </li><br />
<li>- Growth medium</li><br />
<li>- 80% glycerol</li><br />
<li>- centrifuge</li><br />
</ul><br />
<br/><br />
<br />
<i>Protocol:</i><br/><br />
<ol><br />
<li>Take 5 mL bacterial cells from the Erlenmeyer of a freshly grown culture and spin in a 15 mL tube for 10 minutes at 2.000 rpm (Eppendorf centrifuge)</li><br />
<li>Decant the supernatant without disturbing the pellet</li><br />
<li>Pipet on the pellet 0.5 ml of appropriate medium and 0.5 mL 80% glycerol and mix by vortexing and save in -80 °C freezer</li><br />
</ol><br />
<br/><br />
<br />
<br />
<br />
<br />
<a name="P4"> <h1> Restriction enzyme digestion </h1></a><br />
<br/><br />
<i>Materials:</i><br/><br />
<ul><br />
<li>- plasmid DNA or PCR product </li><br />
<li>- restriction enzymes (Roche and BioLabs) </li><br />
<li>- buffer (10x) </li><br />
<li>- H2O</li><br />
<li>water bath at 37 °C</li><br />
<li>- heat block or water bath at 65 °C</li></ul><br/><br />
<i>Protocol:</i><br/><br />
Digestions (cutting plasmid DNA) were performed at the appropriate temperature with the appropriate buffer in the appropriate concentration, according to the supplier. With double restriction, use bigger volume (~ 50 µL) and we found out that adding BSA altered performance greatly.<br/><br/><br />
<br/>Reaction for one sample:<br/><br />
DNA × µL (up to 1,0 µg)<br/><br />
Buffer (10×) × µL (1×))<br/><br />
Restriction enzymes × µL (5 units/µg DNA = 1 µL) )<br/><br />
H2O × µL<br/><br />
tot volume 20-25 µL<br />
<br/><br/><br />
Incubate for (at least) one hour at 37 °C. Inactivate the restriction endonucleases by heat, incubation at 65 °C for 10 minutes and centrifuge shortly.<br/><br />
<br/><br />
<i><u>Used Buffers:</u></i><br/><br />
Buffer H (Roche): 50 mM Tris-HCl, 1 M NaCl, 100 mM MgCl2, 10 mM DTE, pH 7.5 at 37 °C<br/><br />
Buffer M (Roche): 100 mM Tris-HCl, 500 mM NaCI, 100 mM MgCl2, 10 mM DTE, pH 7.5 at 37 °C<br/><br />
Buffer 1 (BioLabs): 10 mM Bis-Tris-Propane-HCl, 10 mM MgCl2, 1 mM DTE,pH 7.0 at 25°C<br/><br />
Buffer 2 (BioLabs): 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTE, pH 7.9 at 25°C<br/><br />
Buffer 3 (BioLabs): 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 1 mM DTE, pH 7.9 at 25°C<br/><br />
Buffer 4 (BioLabs): 50 mM CH3CO2K, 20 mM TAE, 10 mM Mg(CH3COO)2, 1 mM DTE, pH 7.9 at 25°C<br/><br/><br />
Note: some of the restriction enzymes of New England BioLabs required the addition of 100 µg/mL BSA)<br/><br />
<br />
<br><br />
<br />
<br />
<br />
<a name="P5"> <h1>Ligation</h1></a><br />
<br/><br />
<i>Materials:</i><br />
<ul><br />
<li>- digested plasmid DNA or PCR product</li><br />
<li>- T4 ligation buffer (10x) (Fermentas)</li><br />
<li>- T4 ligase (Fermentas)</li><br />
<li>- H2O</li><br />
<li>water bath at 16 °C</li><br />
</ul><br />
<br/><br />
<i>Protocol:</i><br />
Ligations (pasting plasmid DNA) were performed at the appropriate temperature with the appropriate buffer in the appropriate concentration, according to the supplier. We encountered different tactics for ligation. Usually it comes down to keeping it ~16 °C for at least 3 hours.<br />
<br/><br/><br />
<br />
Reaction for one sample:<br/><br />
DNA insert × µL<br/><br />
DNA vector × µL <br/><br />
T4 Ligation buffer (10×) × µL (for 1×)<br/><br />
T4 Ligase 1.0 µL <br/><br />
H2O × µL<br/><br />
tot. volume 10-15 µL<br/><br/><br />
<br />
The final concentration is preferably ~100 ng/µL. Smaller volumes are preferred and when DNA is at low concentration, try to evaporate water using a vacuum. Incubate at 16 °C for at least 3 hours or keep in an ice box in a floatie overnight. In the morning you find eppendorf tubes floating in water which had a temperature gradient overnight. For transformation use circa half of the ligation mix. <br/><br/><br />
<br />
<br />
<br />
<br />
<a name="P6"> <h1>Running a DNA gel</h1></a><br />
<br/><br />
<i>Materials:</i><br />
<ul><br />
<li>- Agarose </li><br />
<li>- TAE 1x </li><br />
<li>- SybrSafe DNA stain</li><br />
<li>- Loading Dye</li><br />
<li>- DNA ladder (Smartladder)</li><br />
<li>- DNA electrophoresis machine</li><br />
</ul><br />
<br/><br />
<i>Protocol:</i><br/><br />
<ol><br />
<li> Dissolve agarose (w/v 0.6% for separating long DNA pieces (>10 kbp), 1% for separating shorter pieces) in 1x TAE by microwaving</li><br />
<li> Close sides of electrophoresis tray (scotch tape works fine) and add comb</li><br />
<li> Let solution cool and add 5 µl Sybrsafe to an empty electrophoresis tray (small gels) 10-12 µl Sybrsafe for larger gels </li><br />
<li> Pour gel until a height of ~0.5 cm. Mix and remove bubbles with pipet tip (fast! It hardens quickly)</li><br />
<li> Put tray into electrophoresis casing and add TAE until a small layer above the gel can be seen. Remove comb</li><br />
<li>Add 1 µl loading dye to 5 µl sample, mix and load in the gel. Also add 5 µl smartladder for your reference</li><br />
<li>Run gel on 80 V (long run)- 110 V (short run, mostly for a ‘fast check’) for ~40-60 minutes, dependant of gel size, separation acquisition and voltage.</li><br />
</ol><br/><br />
For information about the smartladder, <a href=" https://secure.eurogentec.com/product/research-smartladder.html">Smartladder specifications</a>.<br />
<br/><br/><br />
<br />
<br />
<br />
<a name="P7"> <h1>PCR</h1></a><br />
<br/><br />
<i>Materials:</i><br/><br />
For PCR with minimal errors, pFX polymerase is used:<br />
<ul><br />
<li>Pfx polymerase (Invitrogen) </li><li><br />
10x Pfx Buffer (Invitrogen) </li><li><br />
enhancer (Invitrogen) </li><li><br />
50 mM MgSO4 (Invitrogen) </li><li><br />
10 mM dNTPs </li><br />
</ul><br />
<br/><br />
<br />
For PCR for checks of length, Taq polymerase is used, provided in Qiagen mastermix:<br/><br />
<ul><li>Mastermix </li></ul><br />
<br/><br />
Always needed:<br/><br />
<ul><br />
<li>primer solutions 5 mol/mL</li><li><br />
template DNA (plasmid at 50 pg – 1 ng/µL), or plate with colonies</li><li><br />
PCR machine</li><br />
</ul><br />
<br/><br/><br />
<br />
<br />
<i>Protocol:</i><br/><br />
First make sure that there is a PCR machine available for you. Take the solutions from the freezer and thaw them on ice.<br/><br/><br />
Preparation of reaction mixture:<br />
<ol><li>Gently vortex and briefly centrifuge all solutions after thawing</li><br />
<li>Keep solutions on ice</li><br />
<li>Add to a thin walled PCR tube, on ice the desired reaction mixture listed below.</li><br />
<li>for PCR on colonies: prick a sterile toothpick into a colony, dip it into a PCR tube and put it in 15 mL culture tube containing growth media to grow overnight for direct culturing positives.</li><br />
<li>Make sure you keep everything cool until it enters the preheated PCR machine</li></ol><br />
<br/><br />
<br />
<br />
<hr><br />
<br />
<div class=WordSection1><br />
<br />
<p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>For <span<br />
class=SpellE>Pfx</span> PCR the reaction mixture is:<o:p></o:p></span></p><br />
<br />
<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0<br />
style='margin-left:18.0pt;border-collapse:collapse;border:none;mso-border-alt:<br />
solid windowtext .5pt;mso-yfti-tbllook:480;mso-padding-alt:0cm 5.4pt 0cm 5.4pt;<br />
mso-border-insideh:.5pt solid windowtext;mso-border-insidev:.5pt solid windowtext'><br />
<tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'><br />
<td width=196 valign=top style='width:147.15pt;border:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><b>Component<o:p></o:p></b></p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border:solid windowtext 1.5pt;<br />
border-left:none;mso-border-left-alt:solid windowtext 1.5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><b>Sample<o:p></o:p></b></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:1'><br />
<td width=196 valign=top style='width:147.15pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext 1.5pt;mso-border-alt:<br />
solid windowtext 1.5pt;mso-border-bottom-alt:solid windowtext .5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><st1:metricconverter ProductID="10 mM" w:st="on">10 <span<br />
class=SpellE>mM</span></st1:metricconverter> <span class=SpellE>dNTPs</span></p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext 1.5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext 1.5pt;mso-border-bottom-alt:solid windowtext .5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'>1.5 <span<br />
class=SpellE>&#956;L</span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:2'><br />
<td width=196 valign=top style='width:147.15pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Enhancer</span> (<span class=SpellE>Invitrogen</span>)</p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;<br />
mso-border-right-alt:1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'>5.0 <span<br />
class=SpellE>&#956;L</span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:3'><br />
<td width=196 valign=top style='width:147.15pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal style='line-height:normal'>10x <span class=SpellE>Pfx</span><br />
Buffer (<span class=SpellE>Invitrogen</span>)</p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;<br />
mso-border-right-alt:1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'>5.0 <span<br />
class=SpellE>&#956;L</span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:4'><br />
<td width=196 valign=top style='width:147.15pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal style='line-height:normal'><st1:metricconverter<br />
ProductID="50 mM" w:st="on"><span lang=DE style='mso-ansi-language:DE'>50 <span<br />
class=SpellE>mM</span></span></st1:metricconverter><span lang=DE<br />
style='mso-ansi-language:DE'> MgSO<sub>4</sub> (<span class=SpellE>Invitrogen</span>)<br />
<o:p></o:p></span></p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;<br />
mso-border-right-alt:1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'>1.0 <span<br />
class=SpellE>&#956;L</span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:5'><br />
<td width=196 valign=top style='width:147.15pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>Primer 1 </p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;<br />
mso-border-right-alt:1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'>3.0 <span<br />
class=SpellE>&#956;L</span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:6'><br />
<td width=196 valign=top style='width:147.15pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>Primer 2 </p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;<br />
mso-border-right-alt:1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'>3.0 <span<br />
class=SpellE>&#956;L</span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:7'><br />
<td width=196 valign=top style='width:147.15pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal style='line-height:normal'><span class=SpellE>Pfx</span> <span<br />
class=SpellE>polymerase</span> (<span class=SpellE>Invitrogen</span>)</p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;<br />
mso-border-right-alt:1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'>0.6 <span<br />
class=SpellE>&#956;L</span> </p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:8'><br />
<td width=196 valign=top style='width:147.15pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>DNA <span class=SpellE>template</span></p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;<br />
mso-border-right-alt:1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'>1.0 <span<br />
class=SpellE>&#956;L</span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:9;mso-yfti-lastrow:yes'><br />
<td width=196 valign=top style='width:147.15pt;border:solid windowtext 1.5pt;<br />
border-top:none;mso-border-top-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>H<sub>2</sub>O </p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'>29.9 <span<br />
class=SpellE>&#956;L</span></p><br />
</td><br />
</tr><br />
</table><br />
<br/><br />
<p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>PCR program pFX : <br />
</span></p><br />
<br />
<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0 width=414<br />
style='width:310.55pt;margin-left:18.0pt;border-collapse:collapse;border:none;<br />
mso-border-alt:solid windowtext .5pt;mso-yfti-tbllook:480;mso-padding-alt:<br />
0cm 5.4pt 0cm 5.4pt;mso-border-insideh:.5pt solid windowtext;mso-border-insidev:<br />
.5pt solid windowtext'><br />
<tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'><br />
<td width=137 valign=top style='width:102.65pt;border:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><b>Step<o:p></o:p></b></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:solid windowtext 1.5pt;<br />
border-left:none;border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.0pt;<br />
mso-border-left-alt:solid windowtext 1.5pt;mso-border-alt:solid windowtext 1.5pt;<br />
mso-border-right-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE><b>Annealing</b></span><b><o:p></o:p></b></p><br />
<p class=MsoNormal><span class=SpellE><b>Temperature</b></span><b><o:p></o:p></b></p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:solid windowtext 1.5pt;<br />
border-left:none;border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.0pt;<br />
mso-border-left-alt:solid windowtext .5pt;mso-border-top-alt:1.5pt;<br />
mso-border-left-alt:.5pt;mso-border-bottom-alt:1.5pt;mso-border-right-alt:<br />
.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><b>Time, <o:p></o:p></b></p><br />
<p class=MsoNormal><b>min:sec<o:p></o:p></b></p><br />
</td><br />
<td width=91 valign=top style='width:68.0pt;border:solid windowtext 1.5pt;<br />
border-left:none;mso-border-left-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE><b>Number</b></span><b> of <span<br />
class=SpellE>cycles</span><o:p></o:p></b></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:1'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext 1.5pt;mso-border-alt:<br />
solid windowtext 1.5pt;mso-border-bottom-alt:solid windowtext .5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Initial</span> <span class=SpellE>denaturation</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext 1.5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:1.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:<br />
.5pt;mso-border-right-alt:.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>95 °C</p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext 1.5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-top-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>2:00</p><br />
</td><br />
<td width=91 rowspan=3 valign=top style='width:68.0pt;border-top:none;<br />
border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext 1.5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-top-alt:1.5pt;mso-border-left-alt:.5pt;mso-border-bottom-alt:.5pt;<br />
mso-border-right-alt:1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<p class=MsoNormal>1</p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:2'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Annealing</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span style='mso-spacerun:yes'> </span>X °C *</p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>1:00</p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:3'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Extension</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><st1:metricconverter ProductID="68&#65456;C" w:st="on">68°C</st1:metricconverter></p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>1:00-2:00 </p><br />
<p class=MsoNormal>(1 min/<span class=SpellE>kb</span>)</p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:4;page-break-inside:avoid'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Denaturation</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><st1:metricconverter ProductID="95 &#65456;C" w:st="on">95<br />
°C</st1:metricconverter></p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>1:00</p><br />
</td><br />
<td width=91 rowspan=3 valign=top style='width:68.0pt;border-top:none;<br />
border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-right-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<p class=MTDisplayEquation style='tab-stops:35.4pt'><span lang=EN-US<br />
style='font-size:11.0pt;mso-bidi-font-size:12.0pt;font-family:"Calibri","sans-serif"'>25<o:p></o:p></span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:5;page-break-inside:avoid'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Annealing</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span style='mso-spacerun:yes'> </span>X °C *</p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>1:00</p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:6;page-break-inside:avoid'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Extension</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><st1:metricconverter ProductID="68 &#65456;C" w:st="on">68<br />
°C</st1:metricconverter></p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>1:00-2:00 </p><br />
<p class=MsoNormal>(1 min/<span class=SpellE>kb</span>)</p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:7;mso-yfti-lastrow:yes;page-break-inside:avoid'><br />
<td width=137 valign=top style='width:102.65pt;border:solid windowtext 1.5pt;<br />
border-top:none;mso-border-top-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Final</span> <span class=SpellE>Extension</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:1.5pt;<br />
mso-border-right-alt:.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><st1:metricconverter ProductID="68 &#65456;C" w:st="on">68<br />
°C</st1:metricconverter></p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-bottom-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>10:00</p><br />
</td><br />
<td width=91 valign=top style='width:68.0pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>1</p><br />
</td><br />
</tr><br />
</table><br />
<br/><br />
<br />
<p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>For <span<br />
class=SpellE>Taq</span> PCR the reaction mixture is:<o:p></o:p></span></p><br />
<br />
<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0<br />
style='margin-left:18.0pt;border-collapse:collapse;border:none;mso-border-alt:<br />
solid windowtext .5pt;mso-yfti-tbllook:480;mso-padding-alt:0cm 5.4pt 0cm 5.4pt;<br />
mso-border-insideh:.5pt solid windowtext;mso-border-insidev:.5pt solid windowtext'><br />
<tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'><br />
<td width=196 valign=top style='width:147.15pt;border:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><b>Component<o:p></o:p></b></p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border:solid windowtext 1.5pt;<br />
border-left:none;mso-border-left-alt:solid windowtext 1.5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><b>Sample<o:p></o:p></b></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:1'><br />
<td width=196 valign=top style='width:147.15pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext 1.5pt;mso-border-alt:<br />
solid windowtext 1.5pt;mso-border-bottom-alt:solid windowtext .5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE><span lang=EN-US style='mso-ansi-language:<br />
EN-US'>Taq</span></span><span lang=EN-US style='mso-ansi-language:EN-US'> PCR<br />
Master Mix (<span class=SpellE>Qiagen</span><o:p></o:p></span></p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext 1.5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext 1.5pt;mso-border-bottom-alt:solid windowtext .5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'>12.5 <span<br />
class=SpellE>&#956;L</span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:2'><br />
<td width=196 valign=top style='width:147.15pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>Primer 1</p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;<br />
mso-border-right-alt:1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'><span lang=EN-US<br />
style='mso-ansi-language:EN-US'>1.5 </span>&#956;<span lang=EN-US<br />
style='mso-ansi-language:EN-US'>L<o:p></o:p></span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:3'><br />
<td width=196 valign=top style='width:147.15pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>Primer 2<o:p></o:p></span></p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;<br />
mso-border-right-alt:1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'><span lang=EN-US<br />
style='mso-ansi-language:EN-US'>1.5 </span>&#956;<span lang=EN-US<br />
style='mso-ansi-language:EN-US'>L<o:p></o:p></span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:4'><br />
<td width=196 valign=top style='width:147.15pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>Template<br />
DNA (or pick a colony)<o:p></o:p></span></p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;<br />
mso-border-right-alt:1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'><span lang=EN-US<br />
style='mso-ansi-language:EN-US'>1 <span class=SpellE>&#956;L</span><o:p></o:p></span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:5;mso-yfti-lastrow:yes'><br />
<td width=196 valign=top style='width:147.15pt;border:solid windowtext 1.5pt;<br />
border-top:none;mso-border-top-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'>H<sub>2</sub>O<br />
<o:p></o:p></span></p><br />
</td><br />
<td width=67 valign=top style='width:50.1pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal align=right style='text-align:right'><span lang=EN-US<br />
style='mso-ansi-language:EN-US'>8.5 </span>&#956;<span lang=EN-US<br />
style='mso-ansi-language:EN-US'>L<o:p></o:p></span></p><br />
</td><br />
</tr><br />
</table><br />
<br />
<br/><br />
<br />
<p class=MsoNormal>PCR program <span class=SpellE>Taq</span> <span<br />
class=SpellE>polymerase</span><span style='mso-bidi-font-weight:bold'>:</span></p><br />
<br />
<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0 width=414<br />
style='width:310.55pt;margin-left:18.0pt;border-collapse:collapse;border:none;<br />
mso-border-alt:solid windowtext .5pt;mso-yfti-tbllook:480;mso-padding-alt:<br />
0cm 5.4pt 0cm 5.4pt;mso-border-insideh:.5pt solid windowtext;mso-border-insidev:<br />
.5pt solid windowtext'><br />
<tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'><br />
<td width=137 valign=top style='width:102.65pt;border:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><b>Step<o:p></o:p></b></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:solid windowtext 1.5pt;<br />
border-left:none;border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.0pt;<br />
mso-border-left-alt:solid windowtext 1.5pt;mso-border-alt:solid windowtext 1.5pt;<br />
mso-border-right-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE><b>Annealing</b></span><b><o:p></o:p></b></p><br />
<p class=MsoNormal><span class=SpellE><b>Temperature</b></span><b><o:p></o:p></b></p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:solid windowtext 1.5pt;<br />
border-left:none;border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.0pt;<br />
mso-border-left-alt:solid windowtext .5pt;mso-border-top-alt:1.5pt;<br />
mso-border-left-alt:.5pt;mso-border-bottom-alt:1.5pt;mso-border-right-alt:<br />
.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><b>Time, <o:p></o:p></b></p><br />
<p class=MsoNormal><b>min:sec<o:p></o:p></b></p><br />
</td><br />
<td width=91 valign=top style='width:68.0pt;border:solid windowtext 1.5pt;<br />
border-left:none;mso-border-left-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE><b>Number</b></span><b> of <span<br />
class=SpellE>cycles</span><o:p></o:p></b></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:1'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext 1.5pt;mso-border-alt:<br />
solid windowtext 1.5pt;mso-border-bottom-alt:solid windowtext .5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Initial</span> <span class=SpellE>denaturation</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext 1.5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:1.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:<br />
.5pt;mso-border-right-alt:.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>94 °C</p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext 1.5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-top-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>10:00</p><br />
</td><br />
<td width=91 rowspan=3 valign=top style='width:68.0pt;border-top:none;<br />
border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext 1.5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-top-alt:1.5pt;mso-border-left-alt:.5pt;mso-border-bottom-alt:.5pt;<br />
mso-border-right-alt:1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<p class=MsoNormal>1</p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:2'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Annealing</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span style='mso-spacerun:yes'> </span>X °C *</p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>0:45</p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:3'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Extension</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>72°C</p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>1:00-2:00 </p><br />
<p class=MsoNormal>(1 min/<span class=SpellE>kb</span>)</p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:4;page-break-inside:avoid'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Denaturation</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><st1:metricconverter ProductID="95 &#65456;C" w:st="on">95<br />
°C</st1:metricconverter></p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>1:00</p><br />
</td><br />
<td width=91 rowspan=3 valign=top style='width:68.0pt;border-top:none;<br />
border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-right-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><o:p>&nbsp;</o:p></p><br />
<p class=MTDisplayEquation style='tab-stops:35.4pt'><span lang=EN-US<br />
style='font-size:11.0pt;mso-bidi-font-size:12.0pt;font-family:"Calibri","sans-serif"'>25<o:p></o:p></span></p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:5;page-break-inside:avoid'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Annealing</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span style='mso-spacerun:yes'> </span>X °C *</p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>1:00</p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:6;page-break-inside:avoid'><br />
<td width=137 valign=top style='width:102.65pt;border-top:none;border-left:<br />
solid windowtext 1.5pt;border-bottom:solid windowtext 1.0pt;border-right:<br />
solid windowtext 1.5pt;mso-border-top-alt:solid windowtext .5pt;mso-border-top-alt:<br />
.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:.5pt;mso-border-right-alt:<br />
1.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:solid;padding:<br />
0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Extension</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>72 °C</p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>1:00-2:00 </p><br />
<p class=MsoNormal>(1 min/<span class=SpellE>kb</span>)</p><br />
</td><br />
</tr><br />
<tr style='mso-yfti-irow:7;mso-yfti-lastrow:yes;page-break-inside:avoid'><br />
<td width=137 valign=top style='width:102.65pt;border:solid windowtext 1.5pt;<br />
border-top:none;mso-border-top-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal><span class=SpellE>Final</span> <span class=SpellE>Extension</span></p><br />
</td><br />
<td width=98 valign=top style='width:73.15pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext 1.5pt;<br />
mso-border-top-alt:.5pt;mso-border-left-alt:1.5pt;mso-border-bottom-alt:1.5pt;<br />
mso-border-right-alt:.5pt;mso-border-color-alt:windowtext;mso-border-style-alt:<br />
solid;padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>72 °C</p><br />
</td><br />
<td width=89 valign=top style='width:66.75pt;border-top:none;border-left:<br />
none;border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.0pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
mso-border-alt:solid windowtext .5pt;mso-border-bottom-alt:solid windowtext 1.5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>10:00</p><br />
</td><br />
<td width=91 valign=top style='width:68.0pt;border-top:none;border-left:none;<br />
border-bottom:solid windowtext 1.5pt;border-right:solid windowtext 1.5pt;<br />
mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;<br />
padding:0cm 5.4pt 0cm 5.4pt'><br />
<p class=MsoNormal>1</p><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<br/><br />
<br />
<p>*Annealing temperature is very dependent on primer. Optimal temperature: 3x G/C + 2x A/T </p><br />
<br />
<br />
<br/><br />
<br />
<a name="P8"> <h1>Gel Extraction</h1></a><br />
<br/><br />
This protocol is based on QIAGEN® Gel Extraction Handbook.<br />
<br/><br/><br />
<i>Materials:</i><br />
<br/><br />
<ul><li>QIAquick columns</li><li><br />
buffer QG</li><li><br />
buffer PE</li><li><br />
isopropanol</li><li><br />
milliQ</li><li><br />
microcentrifuge</li><li><br />
heat block at 50 °C</li><br />
</ul><br />
<br/><br />
<i>Protocol:</i><br />
<ol><br />
<li>Excise the DNA fragment from the agarose gel with a clean, sharp scalpel. Minimize the size of the gel slice by removing extra agarose. <br />
</li><br />
<li><br />
Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg ~ 100 µL). For >2% agarose gels, add 6 volumes of Buffer QG. The maximum amount of gel slice per QIAquick column is 400 mg; for gel slices >400 mg use more than one QIAquick column. <br />
</li><br />
<li><br />
Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2–3 min during the incubation. <br/><br />
IMPORTANT: Solubilize agarose completely. For >2% gels, increase incubation time. <br />
</li><br />
<li><br />
After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 µL of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow. The adsorption of DNA to the QIAquick membrane is efficient only at pH <7.5. Buffer QG contains a pH indicator which is yellow at pH <7.5 and orange or violet at higher pH, allowing easy determination of the optimal pH for DNA binding. <br />
</li><br />
<li><br />
Add 1 gel volume of isopropanol to the sample and mix. This step increases the yield of DNA fragments <500 bp and >4 kb. For DNA fragments between 500 bp and 4 kb, addition of isopropanol has no effect on yield. Do not centrifuge the sample at this stage. <br />
</li><br />
<li><br />
Place a QIAquick spin column in a provided 2 ml collection tube. <br />
</li><br />
<li><br />
To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 min. <br />
The maximum volume of the column reservoir is 800 µL. For sample volumes of more than 800 µL, simply load and spin again. <br />
</li><br />
<li><br />
Discard flow-through and place QIAquick column back in the same collection tube. <br />
</li><br />
<li><br />
To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min. <br />
</li><br />
<li><br />
Discard the flow-through and centrifuge the QIAquick column for an additional 1 min at 10,000 x g (~13,000 rpm). <br />
</li><br />
<li><br />
IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation. <br />
</li><br />
<li><br />
Place QIAquick column into a clean 1.5 ml microcentrifuge tube. <br />
</li><br />
<li><br />
To elute DNA, add 50 µL of Buffer EB (10 mM Tris•Cl, pH 8.5) or H2O to the center of the QIAquick membrane and centrifuge the column for 1 min at maximum speed. Alternatively, for increased DNA concentration, add 30 µl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min. <br/><br />
Important: Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA. The average eluate volume is 48 µL from 50 µL elution buffer volume, and 28 µL from 30 µL. Elution efficiency is dependent on pH. The maximum elution efficiency is achieved between pH 7.0 and 8.5. When using water, make sure that the pH value is within this range, and store DNA at –20°C as DNA may degrade in the absence of a buffering agent. The purified DNA can also be eluted in TE (10 mM Tris•Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.<br />
</li><br />
</ol><br />
<br />
<br/><br/><br />
<br />
<br />
<a name="P11"> <h1>Immunofluorescence Staining</h1></a><br />
<br/><br />
<div class=WordSection1><br />
<br />
<p class=MsoNoSpacing><span lang=EN-US>This protocol is for immunofluorescence staining<br />
of yeast to prepare it for Flow <span class=SpellE>Cytometry</span> or fluorescence<br />
microscopy, and staining the target of the conjugated antibody of choice. The<br />
TU Delft <span class=SpellE>iGEM</span> 2012 team used a <a<br />
href="http://www.cellsignal.com/products/3916.html"><span class=SpellE>flagtag</span><br />
antibody.</a> <span class=GramE>The</span> protocol of the supplier was<br />
improved for yeast staining. <b style='mso-bidi-font-weight:normal'>IMPORTANT:</b>&nbsp;Please<br />
refer to the&nbsp;<b style='mso-bidi-font-weight:normal'>APPLICATIONS</b><span<br />
class=GramE>&nbsp; section</span> on the front page of the datasheet of the<br />
antibody product to determine if this product is validated and approved for use<br />
on cultured cell lines (IF-IC).</span></p><br />
<br />
<p class=MsoNoSpacing><span lang=EN-US style='mso-fareast-language:NL'><o:p>&nbsp;</o:p></span></p><br />
<br />
<p class=MsoNoSpacing><span lang=EN-US style='mso-bidi-font-family:Calibri;<br />
mso-bidi-theme-font:minor-latin;mso-fareast-language:NL'>Materials:<o:p></o:p></span></p><br />
<br />
<p class=MsoNoSpacing><span lang=EN-US style='mso-bidi-font-family:Calibri;<br />
mso-bidi-theme-font:minor-latin;mso-fareast-language:NL'><o:p>&nbsp;</o:p></span></p><br />
<br />
<p class=MsoNoSpacing style='margin-left:36.0pt;text-indent:-18.0pt;mso-list:<br />
l3 level1 lfo13'><![if !supportLists]><span lang=EN-US style='font-family:Symbol;<br />
mso-fareast-font-family:Symbol;mso-bidi-font-family:Symbol;mso-fareast-language:<br />
NL'><span style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span lang=EN-US style='mso-fareast-language:<br />
NL'><a href="http://openwetware.org/wiki/PBS">Phosphate Buffered Saline (PBS)</a>,<br />
adjust pH to 8.0.<o:p></o:p></span></p><br />
<br />
<p class=MsoNoSpacing style='margin-left:36.0pt;text-indent:-18.0pt;mso-list:<br />
l3 level1 lfo13'><![if !supportLists]><span lang=EN-US style='font-family:Symbol;<br />
mso-fareast-font-family:Symbol;mso-bidi-font-family:Symbol;mso-fareast-language:<br />
NL'><span style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span lang=EN-US style='mso-fareast-language:<br />
NL'>Formaldehyde, 16%, <b style='mso-bidi-font-weight:normal'>methanol free</b><o:p></o:p></span></p><br />
<br />
<p class=MsoNoSpacing style='margin-left:36.0pt;text-indent:-18.0pt;mso-list:<br />
l3 level1 lfo13'><![if !supportLists]><span lang=EN-US style='font-family:Symbol;<br />
mso-fareast-font-family:Symbol;mso-bidi-font-family:Symbol;mso-fareast-language:<br />
NL'><span style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span lang=EN-US style='mso-fareast-language:<br />
NL'>Blocking Buffer:&nbsp;(1X PBS / 1 <span class=SpellE>wt</span>% BSA / 0.3%<br />
(v/v) Triton X-100)<o:p></o:p></span></p><br />
<br />
<p class=MsoNoSpacing style='margin-left:36.0pt;text-indent:-18.0pt;mso-list:<br />
l3 level1 lfo13'><![if !supportLists]><span lang=EN-US style='font-family:Symbol;<br />
mso-fareast-font-family:Symbol;mso-bidi-font-family:Symbol;mso-fareast-language:<br />
NL'><span style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span lang=EN-US style='mso-fareast-language:<br />
NL'>Antibody Dilution Buffer&nbsp;(1X PBS / 1 <span class=SpellE>wt</span>% BSA<br />
/ 0.3% (v/v) Triton X-100)<o:p></o:p></span></p><br />
<br />
<p class=MsoNoSpacing style='margin-left:36.0pt;text-indent:-18.0pt;mso-list:<br />
l3 level1 lfo13'><![if !supportLists]><span lang=EN-US style='font-family:Symbol;<br />
mso-fareast-font-family:Symbol;mso-bidi-font-family:Symbol;mso-fareast-language:<br />
NL'><span style='mso-list:Ignore'>·<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span lang=EN-US style='mso-fareast-language:<br />
NL'>Primary conjugated antibody.<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:<br />
22.2pt;background:white'><b><span style='mso-fareast-font-family:"Times New Roman";<br />
mso-bidi-font-family:Calibri;mso-bidi-theme-font:minor-latin;color:#2B6EAE;<br />
mso-ansi-language:NL;mso-fareast-language:NL'><o:p>&nbsp;</o:p></span></b></p><br />
<br />
<p class=MsoNoSpacing><span lang=EN-US style='mso-fareast-language:NL'>Protocol:<o:p></o:p></span></p><br />
<br />
<p class=MsoNoSpacing><span lang=EN-US style='mso-fareast-language:NL'><o:p>&nbsp;</o:p></span></p><br />
<br />
<p class=MsoNoSpacing><span lang=EN-US style='mso-fareast-language:NL'>The<br />
desired culture of cell is taken for staining and the liquid is aspirated by<br />
spinning it down in appropriate tubing <span class=GramE>( e.g</span>. standard<br />
<span class=SpellE>Eppendorf</span> tubes )<span style='mso-spacerun:yes'> <br />
</span>and decanting or pipetting the supernatant.<span<br />
style='mso-spacerun:yes'> </span>Afterwards the container is inspected for a<br />
cell pellet of acceptable size, meaning it must be easy to spot in the<br />
container. After this the fixing of the cells can start:<o:p></o:p></span></p><br />
<br />
<p class=MsoNoSpacing><b><span lang=EN-US style='color:#2B6EAE;mso-fareast-language:<br />
NL'><o:p>&nbsp;</o:p></span></b></p><br />
<br />
<p class=MsoNoSpacing style='margin-left:36.0pt;text-indent:-18.0pt;mso-list:<br />
l9 level1 lfo14'><![if !supportLists]><span lang=EN-US style='mso-bidi-font-family:<br />
Calibri;mso-bidi-theme-font:minor-latin;mso-fareast-language:NL'><span<br />
style='mso-list:Ignore'>1.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span lang=EN-US style='mso-fareast-language:<br />
NL'>Cover cells to a depth of 10–15 mm with 4% formaldehyde in PBS and <span<br />
class=SpellE>resuspend</span> cells.<br/><br />
<b>NOTE:</b>&nbsp;Formaldehyde is toxic, use only in fume hood.<o:p></o:p></span></p><br />
<br />
<p class=MsoNoSpacing style='margin-left:36.0pt;text-indent:-18.0pt;mso-list:<br />
l9 level1 lfo14'><![if !supportLists]><span lang=EN-US style='mso-bidi-font-family:<br />
Calibri;mso-bidi-theme-font:minor-latin;mso-fareast-language:NL'><span<br />
style='mso-list:Ignore'>2.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span lang=EN-US style='mso-fareast-language:<br />
NL'>Allow cells to fix for 15 minutes at room temperature.<o:p></o:p></span></p><br />
<br />
<p class=MsoNoSpacing style='margin-left:36.0pt;text-indent:-18.0pt;mso-list:<br />
l9 level1 lfo14'><![if !supportLists]><span lang=EN-US style='mso-bidi-font-family:<br />
Calibri;mso-bidi-theme-font:minor-latin;mso-fareast-language:NL'><span<br />
style='mso-list:Ignore'>3.<span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
</span></span></span><![endif]><span lang=EN-US style='mso-fareast-language:<br />
NL'>Aspirate fixative and rinse three times in PBS for 5 minutes each before<br />
proceeding to the staining section. <span class=GramE>( Use</span> the same<br />
aspirating techniques as above: Spin, Aspirate, add, <span class=SpellE>resuspend</span>.<br />
)<o:p></o:p></span></p><br />
<br />
<p class=MsoNormal style='margin-top:0cm;margin-right:0cm;margin-bottom:0cm;<br />
margin-left:48.0pt;margin-bottom:.0001pt;line-height:22.2pt;background:white'><span<br />
lang=EN-US style='mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:<br />
Calibri;mso-bidi-theme-font:minor-latin;color:black;mso-fareast-language:NL'><o:p>&nbsp;</o:p></span></p><br />
<br />
<p class=MsoNoSpacing><span lang=EN-US style='mso-fareast-language:NL'>All subsequent<br />
incubations should be carried out at room temperature unless otherwise noted in<br />
a humid light-tight box or covered dish/plate to prevent drying and <span<br />
class=SpellE>fluorochrome</span> fading.<o:p></o:p></span></p><br />
<br />
<p><ol><br />
<br />
<li> Block specimen in Blocking Buffer for 60 minutes.</li><br />
<li> While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer. ( A Cell Signaling technology antibody was used with this protocol )</li><br />
<li> Aspirate blocking solution, apply diluted primary antibody.</li><br />
<li> Incubate overnight at 4°C.</li><br />
<li> Rinse three times in PBS for 5 minutes each.<br />
NOTE: Because one is using primary antibodies directly conjugated with ( Alexa Fluor® ) fluorochromes, secondary antibodies are not needed.</li><br />
<li> For best results, examine specimens immediately using appropriate excitation wavelength. For long-term storage, store slides flat at 4°C protected from light.</li><br />
</ol></p><br />
<br />
<br />
</div><br />
<br />
<br />
<br/><br />
<a name="P12"><h1>DNA Staining</h1></a><br/><br/><br />
<i>Materials</i><br/><br />
<ul><li>Your favorite yeast strain</li><br />
<li>Vybrant DyeCycle Orange from invitrogen</li><br />
<li>PBS</li><br />
<li>37°C heat block</li><br />
</ul><br/><br/><br />
<br />
<i>Protocol</i><br/><br />
<ol><li>Centrifuge cells on a table top centrifuge for 10 s on 13.000 rpm </li><br />
<li>Take of supernatant and add PBS</li><br />
<li>Add 2 µL/mL Vybrant DyeCycle Orange and vortex until homogenuous solution</li><br />
<li>Keep for 30 minutes on 37°C and the cells are stained.</li><br/><br/><br />
</ol><br />
<br />
<a name="P13"><h1>Flow cytometry to look at DNA content</h1></a><br/><br />
<i>Materials</i><br/><br />
<ul><li>Yeast culture</li><br />
<li>Vybrant® DyeCycleTM Orange stain</li><br />
<li>Ligand</li><br />
<li>Flow cytometry instrument</li><br/><br />
</ul><br />
<i>Protocol</i><br/><br />
<ol><br />
<li>Grow yeast cells at 30C overnight and re-inocculate in 10 mL medium the next morning</li><br />
<li>If cells are in exponential growth phase add the ligand (also keep a control without ligand)</li><br />
<li>After incubation stain the cells according to protocol</li><br />
<li>Transfer the stained cells in a flow cytometry tube</li><br />
<li>Analyze the samples on a flow cytometer using 488 nm excitation or 532 nm excitation and orange emission</li><br />
<li>After each measurement flush the tube with PBS buffer</li><br />
</ol><br />
<br/><br />
<br/><br />
<a name="P14"><h1>Flow cytometry to look at EGFP expression</h1></a><br/><br />
<br />
<br />
<i>Materials</i><br/><br />
<ul><br />
<li>Yeast culture</li><br />
<li>Ligand</li><br />
<li>Flow cytometry instrument</li><br />
</ul><br />
<br/><br />
<i>Protocol</i><br/><br />
<ol><li>Grow yeast cells at 30C overnight and re-inocculate in 10 mL medium the next morning</li><br />
<li>If cells are in exponential growth phase add the ligand (also keep a control without ligand)</li><br />
<li>Transfer the cells in a flow cytometry tube</li><br />
<li>Analyze the samples on a flow cytometer using 488 nm excitation and 509 nm emission</li><br />
<li>After each measurement flush the tube with PBS buffer</li><br />
</ol><br />
<br/><br />
<br/><br />
<a name="P15"><h1>Fluorimeter experiment</h1></a><br/><br/><br />
<i>Materials</i><br/><br />
<ul><br />
<li>Yeast culture</li><br />
<li>Ligand</li><br />
<li>Fluorimeter plate reader</li><br />
<li>96 well plate</li><br/><br />
</ul><br />
<i>Protocol</i><br />
<br />
<br/><br />
<ol><li>Grow yeast cells at 30C overnight and re-inocculate the next morning</li><br />
<li>Measure after approx. 3h hours the OD600. Dilute the cells with medium until an OD of approx. 0.1</li><br />
<li>Transfer the cells to the 96 well plate. Don't forget to have a well with only medium.</li><br />
<li>Add the ligand.</li><br />
<li>Analyze the samples in the fluorimeter that is kept at 30C. Use filters with 488 nm excitation and 509 nm emission. At the same time measure the OD600.</li><br />
<li>Calculate the fluoresce per biomass by dewing the fluorescence by the OD600 values</li><br />
</ol><br><br />
<br />
<a name="P17"><h1>Growth rate experiment</h1></a><br/><br/><br />
<i>Materials</i><br/><br />
<ul><br />
<li>Yeast cultures (wt with and without alpha feromones and far1KO with and without alpha feromones)</li><br />
<li>Alpha feromones</li><br />
<li>Spectrophotometer</li><br />
<li>30C incubator</li><br/><br />
</ul><br />
<i>Protocol</i><br />
<br />
<br/><br />
<ol><li>Grow yeast cells at 30C overnight and re-inocculate 20 ml of medium with 500µl of grown cells the next morning</li><br />
<li>Measure OD600 every hour. </li><br />
<li>Add alpha feromones after 3hr</li><br />
<li>Measure OD600every hour, dilute if needed.</li><br />
</ol><br><br />
<br />
<a name="#P18"><h1>Smell activity tests with petri dishes</h1></a><br/><br/><br />
<i>Materials</i><br/><br />
<br />
<ul><br />
<br />
<li>4 x Two yeast cultures with each having six yeast-strains</li><br />
<br />
<li>Ligands </li><br />
<br />
Water 2 x<br />
<br />
Alpha pheromone 1000x diluted 2 x<br />
<br />
Isoamyl acetate 99%<br />
<br />
Methyl phenylacete 99%<br />
<br />
Niacin 0.336 g/100 ml<br />
<br />
Methyl nicotinate 10 mM<br />
<br />
<li>Typhoon fluorescence scanner</li><br />
<br />
<li>8 Resealable (hard) plastic boxes of 10x10x10 cm.</li><br/><br />
<br />
</ul><br />
<br />
<br/><br />
<br />
<br/><br />
<br />
<i>Protocol</i><br />
<br />
<br/><br />
<br />
<ol><li>Grow yeast cells at 30C for two days.</li><br />
<br />
<li>Analyze the samples in the Typhoon fluorescence scanner before starting experiment.</li><br />
<br />
<li>Pore approx. 25 ml of every ligand (except alpha pheromone) in the box after cleaning it with alcohol / autoclaving if possible. </li><br />
<br />
<li>Place two sided tape on the inwards part of the boxlid. Gently but firmly push the petri dish on it. Remove the lid of the dish and put the dish upside down in the box. For alpha pheromone; add solution directly on all yeast strains.</li><br />
<br />
<li>After 3.5 hours remove the dish from the lid.</li><br />
<br />
<li>Analyze the samples in the Typhoon fluorescence scanner. Use filters with 488 nm excitation and 509 nm emission. Set PMT to 300. Orientation of dish to Platen.</li><br />
<br />
</ol><br><br><br />
<br />
<br />
<a name="P16"><h1>Tips&Tricks for working with yeast</h1></a><br />
<br />
<p>Since this is the first time that TU Delfts iGEM team is working with yeast, we faced a lot of small yeast-related 'challenges'. With this page we want to inform you about the basics and the pitfalls of working with yeast. </p><br />
<h3>Basics</h3><br />
<p>Yeast, <i>Saccharomyces cerevisiae</i>, is a simple, unicellular eukaryotic organism. This organism has been used for fermentation and baking for over 4000 years and it is probably the oldest domesticated micro-organism in the human history. An important one too, can you imagine a life without bread and beer? (Even if you can imagine it, it would most definitely be less fun!) <br />
Nowadays the whole genomic sequence of <i>Saccharomyces cerevisiae</i> is known and a lot of genomic tools are available.</p><br />
<h3>Auxotrophy</h3><br />
<p> The main advantage from an engineering perspective is that yeast has Auxotrophic markers. In specific strains genes are knocked out which synthesize essential enzymes in the amino acid synthesis routes. By complementing these deficiencies by adding the necessary gene on your DNA this provides a nice selection procedure. <br />
<h3> PRS huttle vectors</h3> <br />
<p>The name pRS415 gives an indication on the presence of a CEN/ARS replication origin. 0 means yeast<br />
integrative plasmid, 1 means that it also can be used to maintain the plasmid in circular<br />
form.<br />
pRS415 Gives an indication of the auxotrophic marker used.<br />
pRS415 Version number… not really different.<br/><br />
<h3>Chromosomal integration</h3><br />
<p>We encountered a lot of problems with plasmids. Because we wanted our constructs to be universal<br />
(with the idea to make it suitable for ‘fast checking’) we tried maintaining a plasmid. As it turned<br />
out, yeast cells are not eager to maintain a plasmid and with our construct we suspect homologous<br />
recombination occurred. After transformation, a PCR on the transformed plasmid, obtained by<br />
isolation, showed two bands instead of the suspected single band, one being ! Integration of the<br />
plasmid is therefore advised! Checking of this can be quite gruesome optimizing the necessary PCR<br />
reactions on your transformed yeast colonies. Chromosomal isolation can therefore improve the<br />
steps. </p><br/><br />
<h3>Knock-out strains</h3> <br />
<p>European iGEM teams have the advantage to have <a href="http://web.uni-<br />
frankfurt.de/fb15/mikro/euroscarf">Euroscarf</a> available to order strains with knocked out ORFs. The typical<br />
nomenclature is also explained here: <a href="http://web.uni-frankfurt.de/fb15/mikro/euroscarf/<br />
stra_des.html">Euroscarf explanation</a>.</p><br/><br />
<br />
<br />
<br />
<br />
</div></div><br />
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<a href='https://2012.igem.org/Main_Page' target="_blank"><div id='logo_igem2'></div><a/></body></html></div>MarkWeijershttp://2012.igem.org/Team:TU-Delft/receptordesignTeam:TU-Delft/receptordesign2012-10-27T02:58:38Z<p>MarkWeijers: </p>
<hr />
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<h2>Content</h2> <br />
<a href="#P4">Chimeric receptor design</a><br><br />
<a href="#P1">DO IT YOURSELF receptor design: What, Why and How</a><br><br />
<a href="#P2">In silico protocol</a><br><br />
<a href="#P3">Example</a><br><br />
<a href="#P9">References</a><br><br />
<br />
<a name="P4"> <br><h2> Chimeric receptor design</h2> </a> <br />
<p>A major hindrance for functional expression of ORs has been that the receptors did not localize in the membrane or that the downstream coupling of the receptor to the Gα did not work properly. It has been shown that the rat olfactory receptor 17 (R17) that responds to octanal can be functionally expressed in many different cell types, including <i>S. cerevisiae</i> [6]. Earlier research investigated on the question whether the RI7 sequence can be used to functionally express other ORs. Sequence analysis of ORs have shown that the N-termini of the receptor are involved in plasma membrane localization, whereas the C-termini generally define the specificity for G protein interaction [7]. Based on this observations <i> Radhika et al.</i> functionally expressed a chimeric OR with the N-terminus and the C-terminus of the RI7 sequence. A schematic picture is shown in figure 2. In this iGEM project we use the same approach as <i>Radhika et al.</i> by substituting the receptor termini with the RI7 sequences.</p><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/3/36/Chimeric_design.png"height="200" width="350" /><br />
<h6>Schematic overview of the chimeric design of the receptor. Figure adapted from <i>Radhika et al.</i>.</h6><br />
<br />
<a name="P1"> <br><h2> DO YOURSELF receptor design: What, Why and How</h2> </a><br />
<br />
<h3>What?</h3> <br />
Protocol for making protein chimeras with a rat G protein coupled receptor (RI7) and Your Favorite Receptor. The order of the DNA sequence looks like this: RI7-[Your Favorite Receptor]-RI7<br />
<h3>Why?</h3><br />
One of the requirements for a working GPCR is that the receptor should be localized into the outside membrane of yeast cell. By replacing the N-terminal part of Your Favorite Receptor by the N-terminal ends of a receptor that is known to be localized into the outside membrane of Saccharomyces cerevisiae (R17), Your Favorite Receptor (YFR) will also be localized into the membrane. The C-terminal part of a GPCR is the alpha subunit binding region. If this is replaced by the RI7 regions a higher affinity with the alpha subunit can be reached [1].<br />
<h3>How?</h3><br />
With this step-by-step protocol we guide you trough all the in silico designing steps. After this the DNA can be transformed in yeast and you have your own olfactory yeast!<br />
<br/><br />
<br />
<br />
<a name="P2"> <br><h2> In silico protocol</h2> </a><br><br />
<ol><li>What: <b>Get your receptor protein sequence code </b><br><br />
Why: To introduce a new receptor chimera in yeast you should start with a GPCR with at least a known sequence and preferably a known ligand.<br><br />
How: By using earlier research on GPCRs. For example a nice GPCR database is http://senselab.med.yale.edu/OdorDB/. Copy the DNA sequence and the protein sequence in a text file. <br><br />
<li>What: <b>look for the transmembrane regions</b><br><br />
Why: Normally GPCRs have seven transmembrane regions. The N-terminal loop is important for the localization in the membrane and should be replaced by the RI7 N-terminal sequence. The C-terminal region directly after the last transmembrane part codes for the alpha-subunit binding region. If this region is replaced by the RI7 region a higher affinity with the alpha subunit can be reached.<br><br />
How: Go to http://elm.eu.org/, enter the protein sequence code and find protein motifs for Saccharomyces cerevisiae and the original species. Compare the Global domain table.<br />
Ideally it finds seven transmembrane regions that all have approximately the same length (quite a conserved domains are found[2] ). <br />
When this is not the case, investigate the hydrophobicity by a hydrophobicity index (analysis can be done by Matlab Bioinformatics tool, but this less conclusive due to multiple hydrophobicity indexes). <br><br />
<li>What: <b>Remove protein sequences that code for the N-terminal and C-terminal regions</b><br><br />
Why: The sequence that code for the N-terminal loop should be replaced by the RI7 sequence for better membrane localization. The C-terminal region should be replaced for a higher affinity with the alpha subunit.<br><br />
How: Delete the sequence at the N-terminal end directly after the first transmembrane part (when read from N=left to C=right). <br><br />
Delete the C terminus directly after the last transmembrane part, this is the subunit binding region.<br><br />
<li>What: <b>Check what you did so far</b><br><br />
Why: You want to know if you removed the right regions. Do I have receptor with only six transmembrane regions? <br><br />
How: Check with http://elm.eu.org/, enter the protein sequence code and find protein motifs.<br><br />
<li>What: <b>Go from protein sequence to DNA sequence</b><br><br />
Why: For further adaptations it is easier to work with the DNA sequence<br><br />
How: Enter the original full length DNA sequence in http://web.expasy.org/translate/ with the output format “Include nucleotide sequence’. Now you can easily find which nucleotides should be removed. <br><br />
<li>What: <b>Add the RI7 N-terminal DNA code upstream of your DNA sequence and the RI7 C-terminal downstream</b><br><br />
Why: The sequence that code for the N-terminal loop should be replaced by the RI7 sequence for better membrane localization. The C-terminal region should be replaced for a higher affinity with the alpha subunit.<br><br />
How: Go to Biobrick BBa_K775000 in the Registry of Standard Biological Parts, copy the RI7 N-terminal parts and paste this upstream of YFR sequence. Copy also the RI7 N-terminal parts and paste this downstream of YFR sequence.<br><br />
<li>What: <b>Codon optimize the sequence for Saccharomyce cerevisiae</b><br><br />
Why: For better expression of the protein in yeast<br><br />
How: Go to http://www.jcat.de/ and enter the sequence. Also specify the restriction sites that you don’t want to have in the sequence: at least the standard illegal restriction sites: EcoRI, XbaI, PsteI, SpeI.<br><br />
<li>What: <b>add BamHI and NdeI restriction sites and other features</b><br><br />
Why: If you have this two restriction sites you can easily clone your receptor in BBa_K775000 to have a yeast promoter and terminator.<br><br />
How: If you send it for synthesizing just add the nucleotides in your file. If you work with cDNA you can add the restriction sites with PCR. <br><br />
Tip: if you send your sequence for synthesizing you can also add a Kozak sequence for better translation of the protein and A FLAGtag to analyze the localization of the protein into the membrane. <br><br />
<li>What: <b>Final check</b><br><br />
Why: check, check, double check!<br><br />
How: Align your final protein sequence to the original sequence of your receptor and the chimeric receptors (can be done by blasting too). Also look in http://tools.neb.com/NEBcutter2/ for forbidden restriction sites.<br><br />
<li><b>Now you can send you sequence to a synthesizing company or work with isolated DNA. </b><br><br />
</ol><br />
<a name="P3"> <br><h2> Example</h2> </a><br/><br />
<br />
For our example we take ORL2156, found on the olfactory database of the university of Yale. <br />
<br><b>2. The Elm server</b> had the following output on a the protein sequence of ORL2156:<br><br />
<b><h4>Globular domains/ TM domains and signal peptide detected by the SMART server</h4></b><br><br />
<img src="https://static.igem.org/mediawiki/2012/c/c3/Domain.jpg" width="200" align="center"/><br><br />
<br><b>3. When looking at the cDNA code</b> translated by expasy we indicate the yellow as the cut-off region --> <b>Cut out the yellow parts:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/4/47/Cutoutyellowparts.jpg" width="600"/><br><br />
<br><b>5. Now the yellow parts are removed</b> both in the cDNA and protein code, the check is not shown.<br><br><br />
<b>6. Here the sequence of I7 is added.</b> For convenience we made it a picture.<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/3/30/Substitute.jpg" width="600"/><br><br />
<br><b>7. Codon optimization is performed.</b><br><br><br />
<img src="https://static.igem.org/mediawiki/2012/f/ff/Optimalization.jpg" width="400"/><br><br><br />
<b>8. A very important aspect: adding features!</b> This is of course totally dependant on the methods you want to integrate the designed receptor... So we will leave it to your own creativity.<br><br><br />
<b>9. The checking:</b><br><br />
Protein alignment with original receptor:<br><br />
<img src="https://static.igem.org/mediawiki/2012/2/20/Align.jpg" width="500"/><br><br />
Protein alignment with Rat receptor:<br><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Comparison_RI.jpg" width="500"/><br><br />
Region detection by the elm server:<br><br />
<img src="https://static.igem.org/mediawiki/2012/9/96/Laststep_domain.jpg" width="200" align="center"/><br><br />
Looks good! All alpha helices have 22 amino acids through the membrane. Now we can synthesize the construct or design primer to perform PCR on the species used. Also we can run the YASARA program to optimize the binding niche for the specific ligand and then synthesize!<br><br><br />
<br />
<br />
<a name="P9"><h2>References</h2> </a><br />
<h6>[1] Venkat Radhika, Tassula Proikas-Cezanne, Muralidharan Jayaraman, Djamila Onesime, Ji Hee Ha & Danny N Dhanasekaran, Chemical sensing of DNT by engineered olfactory yeast strain, Nature Chemical biology (2007)<br><br />
[2]Janet M. Young et al. Different evolutionary processes shaped the mouse and human olfactory receptor gene families Hum. Mol. Gen. 2002, Vol. 11, No. 5<br/><br />
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<a href='https://2012.igem.org/Main_Page' target="_blank"><div id='logo_igem2'></div><a/></body></html></div>MarkWeijershttp://2012.igem.org/Team:TU-Delft/receptordesignTeam:TU-Delft/receptordesign2012-10-27T02:57:38Z<p>MarkWeijers: </p>
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<h2>Content</h2> <br />
<a href="#P4">Chimeric receptor design</a><br><br />
<a href="#P1">DO IT YOURSELF receptor design: What, Why and How</a><br><br />
<a href="#P2">In silico protocol</a><br><br />
<a href="#P3">Example</a><br><br />
<a href="#P9">References</a><br><br />
<br />
<a name="P4"> <br><h2> Chimeric receptor design</h2> </a> <br />
<p>A major hindrance for functional expression of ORs has been that the receptors did not localize in the membrane or that the downstream coupling of the receptor to the Gα did not work properly. It has been shown that the rat olfactory receptor 17 (R17) that responds to octanal can be functionally expressed in many different cell types, including <i>S. cerevisiae</i> [6]. Earlier research investigated on the question whether the RI7 sequence can be used to functionally express other ORs. Sequence analysis of ORs have shown that the N-termini of the receptor are involved in plasma membrane localization, whereas the C-termini generally define the specificity for G protein interaction [7]. Based on this observations <i> Radhika et al.</i> functionally expressed a chimeric OR with the N-terminus and the C-terminus of the RI7 sequence. A schematic picture is shown in figure 2. In this iGEM project we use the same approach as <i>Radhika et al.</i> by substituting the receptor termini with the RI7 sequences.</p><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/3/36/Chimeric_design.png"height="200" width="350" /><br />
<h6>Schematic overview of the chimeric design of the receptor. Figure adapted from <i>Radhika et al.</i>.</h6><br />
<br />
<a name="P1"> <br><h2> DO YOURSELF receptor design: What, Why and How</h2> </a><br />
<br />
<h3>What?</h3> <br />
Protocol for making protein chimeras with a rat G protein coupled receptor (RI7) and Your Favorite Receptor. The order of the DNA sequence looks like this: RI7-[Your Favorite Receptor]-RI7<br />
<h3>Why?</h3><br />
One of the requirements for a working GPCR is that the receptor should be localized into the outside membrane of yeast cell. By replacing the N-terminal part of Your Favorite Receptor by the N-terminal ends of a receptor that is known to be localized into the outside membrane of Saccharomyces cerevisiae (R17), Your Favorite Receptor (YFR) will also be localized into the membrane. The C-terminal part of a GPCR is the alpha subunit binding region. If this is replaced by the RI7 regions a higher affinity with the alpha subunit can be reached [1].<br />
<h3>How?</h3><br />
With this step-by-step protocol we guide you trough all the in silico designing steps. After this the DNA can be transformed in yeast and you have your own olfactory yeast!<br />
<br/><br />
<br />
<br />
<a name="P2"> <br><h2> In silico protocol</h2> </a><br><br />
<ol><li>What: <b>Get your receptor protein sequence code </b><br><br />
Why: To introduce a new receptor chimera in yeast you should start with a GPCR with at least a known sequence and preferably a known ligand.<br><br />
How: By using earlier research on GPCRs. For example a nice GPCR database is http://senselab.med.yale.edu/OdorDB/. Copy the DNA sequence and the protein sequence in a text file. <br><br />
<li>What: <b>look for the transmembrane regions</b><br><br />
Why: Normally GPCRs have seven transmembrane regions. The N-terminal loop is important for the localization in the membrane and should be replaced by the RI7 N-terminal sequence. The C-terminal region directly after the last transmembrane part codes for the alpha-subunit binding region. If this region is replaced by the RI7 region a higher affinity with the alpha subunit can be reached.<br><br />
How: Go to http://elm.eu.org/, enter the protein sequence code and find protein motifs for Saccharomyces cerevisiae and the original species. Compare the Global domain table.<br />
Ideally it finds seven transmembrane regions that all have approximately the same length (quite a conserved domains are found[2] ). <br />
When this is not the case, investigate the hydrophobicity by a hydrophobicity index (analysis can be done by Matlab Bioinformatics tool, but this less conclusive due to multiple hydrophobicity indexes). <br><br />
<li>What: <b>Remove protein sequences that code for the N-terminal and C-terminal regions</b><br><br />
Why: The sequence that code for the N-terminal loop should be replaced by the RI7 sequence for better membrane localization. The C-terminal region should be replaced for a higher affinity with the alpha subunit.<br><br />
How: Delete the sequence at the N-terminal end directly after the first transmembrane part (when read from N=left to C=right). <br><br />
Delete the C terminus directly after the last transmembrane part, this is the subunit binding region.<br><br />
<li>What: <b>Check what you did so far</b><br><br />
Why: You want to know if you removed the right regions. Do I have receptor with only six transmembrane regions? <br><br />
How: Check with http://elm.eu.org/, enter the protein sequence code and find protein motifs.<br><br />
<li>What: <b>Go from protein sequence to DNA sequence</b><br><br />
Why: For further adaptations it is easier to work with the DNA sequence<br><br />
How: Enter the original full length DNA sequence in http://web.expasy.org/translate/ with the output format “Include nucleotide sequence’. Now you can easily find which nucleotides should be removed. <br><br />
<li>What: <b>Add the RI7 N-terminal DNA code upstream of your DNA sequence and the RI7 C-terminal downstream</b><br><br />
Why: The sequence that code for the N-terminal loop should be replaced by the RI7 sequence for better membrane localization. The C-terminal region should be replaced for a higher affinity with the alpha subunit.<br><br />
How: Go to Biobrick BBa_K775000 in the Registry of Standard Biological Parts, copy the RI7 N-terminal parts and paste this upstream of YFR sequence. Copy also the RI7 N-terminal parts and paste this downstream of YFR sequence.<br><br />
<li>What: <b>Codon optimize the sequence for Saccharomyce cerevisiae</b><br><br />
Why: For better expression of the protein in yeast<br><br />
How: Go to http://www.jcat.de/ and enter the sequence. Also specify the restriction sites that you don’t want to have in the sequence: at least the standard illegal restriction sites: EcoRI, XbaI, PsteI, SpeI.<br><br />
<li>What: <b>add BamHI and NdeI restriction sites and other features</b><br><br />
Why: If you have this two restriction sites you can easily clone your receptor in BBa_K775000 to have a yeast promoter and terminator.<br><br />
How: If you send it for synthesizing just add the nucleotides in your file. If you work with cDNA you can add the restriction sites with PCR. <br><br />
Tip: if you send your sequence for synthesizing you can also add a Kozak sequence for better translation of the protein and A FLAGtag to analyze the localization of the protein into the membrane. <br><br />
<li>What: <b>Final check</b><br><br />
Why: check, check, double check!<br><br />
How: Align your final protein sequence to the original sequence of your receptor and the chimeric receptors (can be done by blasting too). Also look in http://tools.neb.com/NEBcutter2/ for forbidden restriction sites.<br><br />
<li><b>Now you can send you sequence to a synthesizing company or work with isolated DNA. </b><br><br />
</ol><br />
<a name="P3"> <br><h2> Example</h2> </a><br/><br />
<br />
For our example we take ORL2156, found on the olfactory database of the university of Yale. <br />
<br><b>2. The Elm server</b> had the following output on a the protein sequence of ORL2156:<br><br />
<b><h4>Globular domains/ TM domains and signal peptide detected by the SMART server</h4></b><br><br />
<img src="https://static.igem.org/mediawiki/2012/c/c3/Domain.jpg" width="200" align="center"/><br><br />
<br><b>3. When looking at the cDNA code</b> translated by expasy we indicate the yellow as the cut-off region --> <b>Cut out the yellow parts:</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/4/47/Cutoutyellowparts.jpg" width="600"/><br><br />
<br><b>5. Now the yellow parts are removed</b> both in the cDNA and protein code, the check is not shown.<br><br><br />
<b>6. Here the sequence of I7 is added.</b> For convenience we made it a picture.<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/3/30/Substitute.jpg" width="600"/><br><br />
<br><b>7. Codon optimization is performed.</b><br><br><br />
<img src="https://static.igem.org/mediawiki/2012/f/ff/Optimalization.jpg" width="400"/><br><br><br />
<b>8. A very important aspect: adding features!</b> This is of course totally dependant on the methods you want to integrate the designed receptor... So we will leave it to your own creativity.<br><br><br />
<b>9. The checking:</b><br><br />
Protein alignment with original receptor:<br><br />
<img src="https://static.igem.org/mediawiki/2012/2/20/Align.jpg" width="500"/><br><br />
Protein alignment with Rat receptor:<br><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Comparison_RI.jpg" width="500"/><br><br />
Region detection by the elm server:<br><br />
<img src="https://static.igem.org/mediawiki/2012/9/96/Laststep_domain.jpg" width="200" align="center"/><br><br />
Looks good! All 22 amino acids through the membrane. Now we can synthesize the construct or design primer to perform PCR on the species used. Also we can run the YASARA program to optimize the binding niche for the specific ligand and then synthesize!<br><br><br />
<br />
<br />
<a name="P9"><h2>References</h2> </a><br />
<h6>[1] Venkat Radhika, Tassula Proikas-Cezanne, Muralidharan Jayaraman, Djamila Onesime, Ji Hee Ha & Danny N Dhanasekaran, Chemical sensing of DNT by engineered olfactory yeast strain, Nature Chemical biology (2007)<br><br />
[2]Janet M. Young et al. Different evolutionary processes shaped the mouse and human olfactory receptor gene families Hum. Mol. Gen. 2002, Vol. 11, No. 5<br/><br />
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<a href='https://2012.igem.org/Main_Page' target="_blank"><div id='logo_igem2'></div><a/></body></html></div>MarkWeijershttp://2012.igem.org/Team:TU-Delft/receptordesignTeam:TU-Delft/receptordesign2012-10-27T02:55:07Z<p>MarkWeijers: </p>
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<h2>Content</h2> <br />
<a href="#P4">Chimeric receptor design</a><br><br />
<a href="#P1">DO IT YOURSELF receptor design: What, Why and How</a><br><br />
<a href="#P2">In silico protocol</a><br><br />
<a href="#P3">Example</a><br><br />
<a href="#P9">References</a><br><br />
<br />
<a name="P4"> <br><h2> Chimeric receptor design</h2> </a> <br />
<p>A major hindrance for functional expression of ORs has been that the receptors did not localize in the membrane or that the downstream coupling of the receptor to the Gα did not work properly. It has been shown that the rat olfactory receptor 17 (R17) that responds to octanal can be functionally expressed in many different cell types, including <i>S. cerevisiae</i> [6]. Earlier research investigated on the question whether the RI7 sequence can be used to functionally express other ORs. Sequence analysis of ORs have shown that the N-termini of the receptor are involved in plasma membrane localization, whereas the C-termini generally define the specificity for G protein interaction [7]. Based on this observations <i> Radhika et al.</i> functionally expressed a chimeric OR with the N-terminus and the C-terminus of the RI7 sequence. A schematic picture is shown in figure 2. In this iGEM project we use the same approach as <i>Radhika et al.</i> by substituting the receptor termini with the RI7 sequences.</p><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/3/36/Chimeric_design.png"height="200" width="350" /><br />
<h6>Schematic overview of the chimeric design of the receptor. Figure adapted from <i>Radhika et al.</i>.</h6><br />
<br />
<a name="P1"> <br><h2> DO YOURSELF receptor design: What, Why and How</h2> </a><br />
<br />
<h3>What?</h3> <br />
Protocol for making protein chimeras with a rat G protein coupled receptor (RI7) and Your Favorite Receptor. The order of the DNA sequence looks like this: RI7-[Your Favorite Receptor]-RI7<br />
<h3>Why?</h3><br />
One of the requirements for a working GPCR is that the receptor should be localized into the outside membrane of yeast cell. By replacing the N-terminal part of Your Favorite Receptor by the N-terminal ends of a receptor that is known to be localized into the outside membrane of Saccharomyces cerevisiae (R17), Your Favorite Receptor (YFR) will also be localized into the membrane. The C-terminal part of a GPCR is the alpha subunit binding region. If this is replaced by the RI7 regions a higher affinity with the alpha subunit can be reached [1].<br />
<h3>How?</h3><br />
With this step-by-step protocol we guide you trough all the in silico designing steps. After this the DNA can be transformed in yeast and you have your own olfactory yeast!<br />
<br/><br />
<br />
<br />
<a name="P2"> <br><h2> In silico protocol</h2> </a><br><br />
<ol><li>What: <b>Get your receptor protein sequence code </b><br><br />
Why: To introduce a new receptor chimera in yeast you should start with a GPCR with at least a known sequence and preferably a known ligand.<br><br />
How: By using earlier research on GPCRs. For example a nice GPCR database is http://senselab.med.yale.edu/OdorDB/. Copy the DNA sequence and the protein sequence in a text file. <br><br />
<li>What: <b>look for the transmembrane regions</b><br><br />
Why: Normally GPCRs have seven transmembrane regions. The N-terminal loop is important for the localization in the membrane and should be replaced by the RI7 N-terminal sequence. The C-terminal region directly after the last transmembrane part codes for the alpha-subunit binding region. If this region is replaced by the RI7 region a higher affinity with the alpha subunit can be reached.<br><br />
How: Go to http://elm.eu.org/, enter the protein sequence code and find protein motifs for Saccharomyces cerevisiae and the original species. Compare the Global domain table.<br />
Ideally it finds seven transmembrane regions that all have approximately the same length (quite a conserved domains are found[2] ). <br />
When this is not the case, investigate the hydrophobicity by a hydrophobicity index (analysis can be done by Matlab Bioinformatics tool, but this less conclusive due to multiple hydrophobicity indexes). <br><br />
<li>What: <b>Remove protein sequences that code for the N-terminal and C-terminal regions</b><br><br />
Why: The sequence that code for the N-terminal loop should be replaced by the RI7 sequence for better membrane localization. The C-terminal region should be replaced for a higher affinity with the alpha subunit.<br><br />
How: Delete the sequence at the N-terminal end directly after the first transmembrane part (when read from N=left to C=right). <br><br />
Delete the C terminus directly after the last transmembrane part, this is the subunit binding region.<br><br />
<li>What: <b>Check what you did so far</b><br><br />
Why: You want to know if you removed the right regions. Do I have receptor with only six transmembrane regions? <br><br />
How: Check with http://elm.eu.org/, enter the protein sequence code and find protein motifs.<br><br />
<li>What: <b>Go from protein sequence to DNA sequence</b><br><br />
Why: For further adaptations it is easier to work with the DNA sequence<br><br />
How: Enter the original full length DNA sequence in http://web.expasy.org/translate/ with the output format “Include nucleotide sequence’. Now you can easily find which nucleotides should be removed. <br><br />
<li>What: <b>Add the RI7 N-terminal DNA code upstream of your DNA sequence and the RI7 C-terminal downstream</b><br><br />
Why: The sequence that code for the N-terminal loop should be replaced by the RI7 sequence for better membrane localization. The C-terminal region should be replaced for a higher affinity with the alpha subunit.<br><br />
How: Go to Biobrick BBa_K775000 in the Registry of Standard Biological Parts, copy the RI7 N-terminal parts and paste this upstream of YFR sequence. Copy also the RI7 N-terminal parts and paste this downstream of YFR sequence.<br><br />
<li>What: <b>Codon optimize the sequence for Saccharomyce cerevisiae</b><br><br />
Why: For better expression of the protein in yeast<br><br />
How: Go to http://www.jcat.de/ and enter the sequence. Also specify the restriction sites that you don’t want to have in the sequence: at least the standard illegal restriction sites: EcoRI, XbaI, PsteI, SpeI.<br><br />
<li>What: <b>add BamHI and NdeI restriction sites and other features</b><br><br />
Why: If you have this two restriction sites you can easily clone your receptor in BBa_K775000 to have a yeast promoter and terminator.<br><br />
How: If you send it for synthesizing just add the nucleotides in your file. If you work with cDNA you can add the restriction sites with PCR. <br><br />
Tip: if you send your sequence for synthesizing you can also add a Kozak sequence for better translation of the protein and A FLAGtag to analyze the localization of the protein into the membrane. <br><br />
<li>What: <b>Final check</b><br><br />
Why: check, check, double check!<br><br />
How: Align your final protein sequence to the original sequence of your receptor and the chimeric receptors (can be done by blasting too). Also look in http://tools.neb.com/NEBcutter2/ for forbidden restriction sites.<br><br />
<li><b>Now you can send you sequence to a synthesizing company or work with isolated DNA. </b><br><br />
</ol><br />
<a name="P3"> <br><h2> Example</h2> </a><br/><br />
<br />
For our example we take ORL2156, found on the olfactory database of the university of Yale. <br />
<b>2. The Elm server</b> had the following output on a the protein sequence of ORL2156:<br><br />
<b><h4>Globular domains/ TM domains and signal peptide detected by the SMART server</h4></b><br><br />
<img src="https://static.igem.org/mediawiki/2012/c/c3/Domain.jpg" width="200" align="center"/><br><br />
<b>3. When looking at the cDNA code</b> translated by expasy we indicate the yellow as the cut-off region --> <b>Cut out the yellow parts:</b><br />
<img src="https://static.igem.org/mediawiki/2012/4/47/Cutoutyellowparts.jpg" width="600"/><br><br />
<b>5. Now the yellow parts are removed</b> both in the cDNA and protein code, the check is not shown.<br><br><br />
<b>6. Here the sequence of I7 is added.</b> For convenience we made it a picture.<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/3/30/Substitute.jpg" width="600"/><br><br />
<b>7. Codon optimization is performed.</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/f/ff/Optimalization.jpg" width="400"/><br><br><br />
<b>8. A very important aspect: adding features!</b> This is of course totally dependant on the methods you want to integrate the designed receptor... So we will leave it to your own creativity.<br><br><br />
<b>9. The checking:</b><br><br />
Protein alignment with original receptor:<br><br />
<img src="https://static.igem.org/mediawiki/2012/2/20/Align.jpg" width="500"/><br><br />
Protein alignment with Rat receptor:<br><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Comparison_RI.jpg" width="500"/><br><br />
Region detection by the elm server:<br><br />
<img src="https://static.igem.org/mediawiki/2012/9/96/Laststep_domain.jpg" width="200" align="center"/><br><br />
Looks good! All 22 amino acids through the membrane. Now we can synthesize the construct or design primer to perform PCR on the species used. Also we can run the YASARA program to optimize the binding niche for the specific ligand and then synthesize!<br><br><br />
<br />
<br />
<a name="P9"><h2>References</h2> </a><br />
<h6>[1] Venkat Radhika, Tassula Proikas-Cezanne, Muralidharan Jayaraman, Djamila Onesime, Ji Hee Ha & Danny N Dhanasekaran, Chemical sensing of DNT by engineered olfactory yeast strain, Nature Chemical biology (2007)<br><br />
[2]Janet M. Young et al. Different evolutionary processes shaped the mouse and human olfactory receptor gene families Hum. Mol. Gen. 2002, Vol. 11, No. 5<br/><br />
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<a href='https://2012.igem.org/Main_Page' target="_blank"><div id='logo_igem2'></div><a/></body></html></div>MarkWeijershttp://2012.igem.org/Team:TU-Delft/receptordesignTeam:TU-Delft/receptordesign2012-10-27T02:51:15Z<p>MarkWeijers: </p>
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<h2>Content</h2> <br />
<a href="#P4">Chimeric receptor design</a><br><br />
<a href="#P1">DO IT YOURSELF receptor design: What, Why and How</a><br><br />
<a href="#P2">In silico protocol</a><br><br />
<a href="#P3">Example</a><br><br />
<a href="#P9">References</a><br><br />
<br />
<a name="P4"> <br><h2> Chimeric receptor design</h2> </a> <br />
<p>A major hindrance for functional expression of ORs has been that the receptors did not localize in the membrane or that the downstream coupling of the receptor to the Gα did not work properly. It has been shown that the rat olfactory receptor 17 (R17) that responds to octanal can be functionally expressed in many different cell types, including <i>S. cerevisiae</i> [6]. Earlier research investigated on the question whether the RI7 sequence can be used to functionally express other ORs. Sequence analysis of ORs have shown that the N-termini of the receptor are involved in plasma membrane localization, whereas the C-termini generally define the specificity for G protein interaction [7]. Based on this observations <i> Radhika et al.</i> functionally expressed a chimeric OR with the N-terminus and the C-terminus of the RI7 sequence. A schematic picture is shown in figure 2. In this iGEM project we use the same approach as <i>Radhika et al.</i> by substituting the receptor termini with the RI7 sequences.</p><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/3/36/Chimeric_design.png"height="200" width="350" /><br />
<h6>Schematic overview of the chimeric design of the receptor. Figure adapted from <i>Radhika et al.</i>.</h6><br />
<br />
<a name="P1"> <br><h2> DO YOURSELF receptor design: What, Why and How</h2> </a><br />
<br />
<h3>What?</h3> <br />
Protocol for making protein chimeras with a rat G protein coupled receptor (RI7) and Your Favorite Receptor. The order of the DNA sequence looks like this: RI7-[Your Favorite Receptor]-RI7<br />
<h3>Why?</h3><br />
One of the requirements for a working GPCR is that the receptor should be localized into the outside membrane of yeast cell. By replacing the N-terminal part of Your Favorite Receptor by the N-terminal ends of a receptor that is known to be localized into the outside membrane of Saccharomyces cerevisiae (R17), Your Favorite Receptor (YFR) will also be localized into the membrane. The C-terminal part of a GPCR is the alpha subunit binding region. If this is replaced by the RI7 regions a higher affinity with the alpha subunit can be reached [1].<br />
<h3>How?</h3><br />
With this step-by-step protocol we guide you trough all the in silico designing steps. After this the DNA can be transformed in yeast and you have your own olfactory yeast!<br />
<br/><br />
<br />
<br />
<a name="P2"> <br><h2> In silico protocol</h2> </a><br><br />
<ol><li>What: <b>Get your receptor protein sequence code </b><br><br />
Why: To introduce a new receptor chimera in yeast you should start with a GPCR with at least a known sequence and preferably a known ligand.<br><br />
How: By using earlier research on GPCRs. For example a nice GPCR database is http://senselab.med.yale.edu/OdorDB/. Copy the DNA sequence and the protein sequence in a text file. <br><br />
<li>What: <b>look for the transmembrane regions</b><br><br />
Why: Normally GPCRs have seven transmembrane regions. The N-terminal loop is important for the localization in the membrane and should be replaced by the RI7 N-terminal sequence. The C-terminal region directly after the last transmembrane part codes for the alpha-subunit binding region. If this region is replaced by the RI7 region a higher affinity with the alpha subunit can be reached.<br><br />
How: Go to http://elm.eu.org/, enter the protein sequence code and find protein motifs for Saccharomyces cerevisiae and the original species. Compare the Global domain table.<br />
Ideally it finds seven transmembrane regions that all have approximately the same length (quite a conserved domains are found[2] ). <br />
When this is not the case, investigate the hydrophobicity by a hydrophobicity index (analysis can be done by Matlab Bioinformatics tool, but this less conclusive due to multiple hydrophobicity indexes). <br><br />
<li>What: <b>Remove protein sequences that code for the N-terminal and C-terminal regions</b><br><br />
Why: The sequence that code for the N-terminal loop should be replaced by the RI7 sequence for better membrane localization. The C-terminal region should be replaced for a higher affinity with the alpha subunit.<br><br />
How: Delete the sequence at the N-terminal end directly after the first transmembrane part (when read from N=left to C=right). <br><br />
Delete the C terminus directly after the last transmembrane part, this is the subunit binding region.<br><br />
<li>What: <b>Check what you did so far</b><br><br />
Why: You want to know if you removed the right regions. Do I have receptor with only six transmembrane regions? <br><br />
How: Check with http://elm.eu.org/, enter the protein sequence code and find protein motifs.<br><br />
<li>What: <b>Go from protein sequence to DNA sequence</b><br><br />
Why: For further adaptations it is easier to work with the DNA sequence<br><br />
How: Enter the original full length DNA sequence in http://web.expasy.org/translate/ with the output format “Include nucleotide sequence’. Now you can easily find which nucleotides should be removed. <br><br />
<li>What: <b>Add the RI7 N-terminal DNA code upstream of your DNA sequence and the RI7 C-terminal downstream</b><br><br />
Why: The sequence that code for the N-terminal loop should be replaced by the RI7 sequence for better membrane localization. The C-terminal region should be replaced for a higher affinity with the alpha subunit.<br><br />
How: Go to Biobrick BBa_K775000 in the Registry of Standard Biological Parts, copy the RI7 N-terminal parts and paste this upstream of YFR sequence. Copy also the RI7 N-terminal parts and paste this downstream of YFR sequence.<br><br />
<li>What: <b>Codon optimize the sequence for Saccharomyce cerevisiae</b><br><br />
Why: For better expression of the protein in yeast<br><br />
How: Go to http://www.jcat.de/ and enter the sequence. Also specify the restriction sites that you don’t want to have in the sequence: at least the standard illegal restriction sites: EcoRI, XbaI, PsteI, SpeI.<br><br />
<li>What: <b>add BamHI and NdeI restriction sites and other features</b><br><br />
Why: If you have this two restriction sites you can easily clone your receptor in BBa_K775000 to have a yeast promoter and terminator.<br><br />
How: If you send it for synthesizing just add the nucleotides in your file. If you work with cDNA you can add the restriction sites with PCR. <br><br />
Tip: if you send your sequence for synthesizing you can also add a Kozak sequence for better translation of the protein and A FLAGtag to analyze the localization of the protein into the membrane. <br><br />
<li>What: <b>Final check</b><br><br />
Why: check, check, double check!<br><br />
How: Align your final protein sequence to the original sequence of your receptor and the chimeric receptors (can be done by blasting too). Also look in http://tools.neb.com/NEBcutter2/ for forbidden restriction sites.<br><br />
<li><b>Now you can send you sequence to a synthesizing company or work with isolated DNA. </b><br><br />
</ol><br />
<a name="P3"> <br><h2> Example</h2> </a><br/><br />
<br />
For our example we take ORL2156, found on the olfactory database of the university of Yale. <br />
2. The Elm server had the following output on a the protein sequence of ORL2156:<br><br />
<b>Globular domains/ TM domains and signal peptide detected by the SMART server</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/c/c3/Domain.jpg" width="200" align="center"/><br><br />
3. When looking at the cDNA code translated by expasy we indicate the yellow as the cut-off region --> Cut out the yellow parts:<br />
<img src="https://static.igem.org/mediawiki/2012/4/47/Cutoutyellowparts.jpg" width="600"/><br><br />
5. Now the yellow parts are removed both in the cDNA and protein code, the check is not shown.<br><br><br />
6. Here the sequence of I7 is added. For convenience we made it a picture.<br><br><br />
<img src="https://static.igem.org/mediawiki/2012/3/30/Substitute.jpg" width="600"/><br><br />
7. Codon optimization is performed.<br><br />
<img src="https://static.igem.org/mediawiki/2012/f/ff/Optimalization.jpg" width="400"/><br><br><br />
8. A very important aspect: adding features! This is of course totally dependant on the methods you want to integrate the designed receptor... So we will leave it to your own creativity.<br><br><br />
9. The checking:<br />
Protein alignment with original receptor:<br><br />
<img src="https://static.igem.org/mediawiki/2012/2/20/Align.jpg" width="500"/><br><br />
Protein alignment with Rat receptor:<br><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Comparison_RI.jpg" width="500"/><br><br />
Region detection by the elm server:<br><br />
<img src="https://static.igem.org/mediawiki/2012/9/96/Laststep_domain.jpg" width="200" align="center"/><br><br />
Looks good! All 22 amino acids through the membrane. Now we can synthesize the construct or design primer to perform PCR on the species used. Also we can run the YASARA program to optimize the binding niche for the specific ligand and then synthesize!<br><br><br />
<br />
<br />
<a name="P9"><h2>References</h2> </a><br />
<h6>[1] Venkat Radhika, Tassula Proikas-Cezanne, Muralidharan Jayaraman, Djamila Onesime, Ji Hee Ha & Danny N Dhanasekaran, Chemical sensing of DNT by engineered olfactory yeast strain, Nature Chemical biology (2007)<br><br />
[2]Janet M. Young et al. Different evolutionary processes shaped the mouse and human olfactory receptor gene families Hum. Mol. Gen. 2002, Vol. 11, No. 5<br/><br />
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<a href='https://2012.igem.org/Main_Page' target="_blank"><div id='logo_igem2'></div><a/></body></html></div>MarkWeijershttp://2012.igem.org/Team:TU-Delft/receptordesignTeam:TU-Delft/receptordesign2012-10-27T02:49:14Z<p>MarkWeijers: </p>
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<br />
<h2>Content</h2> <br />
<a href="#P4">Chimeric receptor design</a><br><br />
<a href="#P1">DO IT YOURSELF receptor design: What, Why and How</a><br><br />
<a href="#P2">In silico protocol</a><br><br />
<a href="#P3">Example</a><br><br />
<a href="#P9">References</a><br><br />
<br />
<a name="P4"> <br><h2> Chimeric receptor design</h2> </a> <br />
<p>A major hindrance for functional expression of ORs has been that the receptors did not localize in the membrane or that the downstream coupling of the receptor to the Gα did not work properly. It has been shown that the rat olfactory receptor 17 (R17) that responds to octanal can be functionally expressed in many different cell types, including <i>S. cerevisiae</i> [6]. Earlier research investigated on the question whether the RI7 sequence can be used to functionally express other ORs. Sequence analysis of ORs have shown that the N-termini of the receptor are involved in plasma membrane localization, whereas the C-termini generally define the specificity for G protein interaction [7]. Based on this observations <i> Radhika et al.</i> functionally expressed a chimeric OR with the N-terminus and the C-terminus of the RI7 sequence. A schematic picture is shown in figure 2. In this iGEM project we use the same approach as <i>Radhika et al.</i> by substituting the receptor termini with the RI7 sequences.</p><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/3/36/Chimeric_design.png"height="200" width="350" /><br />
<h6>Schematic overview of the chimeric design of the receptor. Figure adapted from <i>Radhika et al.</i>.</h6><br />
<br />
<a name="P1"> <br><h2> DO YOURSELF receptor design: What, Why and How</h2> </a><br />
<br />
<h3>What?</h3> <br />
Protocol for making protein chimeras with a rat G protein coupled receptor (RI7) and Your Favorite Receptor. The order of the DNA sequence looks like this: RI7-[Your Favorite Receptor]-RI7<br />
<h3>Why?</h3><br />
One of the requirements for a working GPCR is that the receptor should be localized into the outside membrane of yeast cell. By replacing the N-terminal part of Your Favorite Receptor by the N-terminal ends of a receptor that is known to be localized into the outside membrane of Saccharomyces cerevisiae (R17), Your Favorite Receptor (YFR) will also be localized into the membrane. The C-terminal part of a GPCR is the alpha subunit binding region. If this is replaced by the RI7 regions a higher affinity with the alpha subunit can be reached [1].<br />
<h3>How?</h3><br />
With this step-by-step protocol we guide you trough all the in silico designing steps. After this the DNA can be transformed in yeast and you have your own olfactory yeast!<br />
<br/><br />
<br />
<br />
<a name="P2"> <br><h2> In silico protocol</h2> </a><br><br />
<ol><li>What: <b>Get your receptor protein sequence code </b><br><br />
Why: To introduce a new receptor chimera in yeast you should start with a GPCR with at least a known sequence and preferably a known ligand.<br><br />
How: By using earlier research on GPCRs. For example a nice GPCR database is http://senselab.med.yale.edu/OdorDB/. Copy the DNA sequence and the protein sequence in a text file. <br><br />
<li>What: <b>look for the transmembrane regions</b><br><br />
Why: Normally GPCRs have seven transmembrane regions. The N-terminal loop is important for the localization in the membrane and should be replaced by the RI7 N-terminal sequence. The C-terminal region directly after the last transmembrane part codes for the alpha-subunit binding region. If this region is replaced by the RI7 region a higher affinity with the alpha subunit can be reached.<br><br />
How: Go to http://elm.eu.org/, enter the protein sequence code and find protein motifs for Saccharomyces cerevisiae and the original species. Compare the Global domain table.<br />
Ideally it finds seven transmembrane regions that all have approximately the same length (quite a conserved domains are found[2] ). <br />
When this is not the case, investigate the hydrophobicity by a hydrophobicity index (analysis can be done by Matlab Bioinformatics tool, but this less conclusive due to multiple hydrophobicity indexes). <br><br />
<li>What: <b>Remove protein sequences that code for the N-terminal and C-terminal regions</b><br><br />
Why: The sequence that code for the N-terminal loop should be replaced by the RI7 sequence for better membrane localization. The C-terminal region should be replaced for a higher affinity with the alpha subunit.<br><br />
How: Delete the sequence at the N-terminal end directly after the first transmembrane part (when read from N=left to C=right). <br><br />
Delete the C terminus directly after the last transmembrane part, this is the subunit binding region.<br><br />
<li>What: <b>Check what you did so far</b><br><br />
Why: You want to know if you removed the right regions. Do I have receptor with only six transmembrane regions? <br><br />
How: Check with http://elm.eu.org/, enter the protein sequence code and find protein motifs.<br><br />
<li>What: <b>Go from protein sequence to DNA sequence</b><br><br />
Why: For further adaptations it is easier to work with the DNA sequence<br><br />
How: Enter the original full length DNA sequence in http://web.expasy.org/translate/ with the output format “Include nucleotide sequence’. Now you can easily find which nucleotides should be removed. <br><br />
<li>What: <b>Add the RI7 N-terminal DNA code upstream of your DNA sequence and the RI7 C-terminal downstream</b><br><br />
Why: The sequence that code for the N-terminal loop should be replaced by the RI7 sequence for better membrane localization. The C-terminal region should be replaced for a higher affinity with the alpha subunit.<br><br />
How: Go to Biobrick BBa_K775000 in the Registry of Standard Biological Parts, copy the RI7 N-terminal parts and paste this upstream of YFR sequence. Copy also the RI7 N-terminal parts and paste this downstream of YFR sequence.<br><br />
<li>What: <b>Codon optimize the sequence for Saccharomyce cerevisiae</b><br><br />
Why: For better expression of the protein in yeast<br><br />
How: Go to http://www.jcat.de/ and enter the sequence. Also specify the restriction sites that you don’t want to have in the sequence: at least the standard illegal restriction sites: EcoRI, XbaI, PsteI, SpeI.<br><br />
<li>What: <b>add BamHI and NdeI restriction sites and other features</b><br><br />
Why: If you have this two restriction sites you can easily clone your receptor in BBa_K775000 to have a yeast promoter and terminator.<br><br />
How: If you send it for synthesizing just add the nucleotides in your file. If you work with cDNA you can add the restriction sites with PCR. <br><br />
Tip: if you send your sequence for synthesizing you can also add a Kozak sequence for better translation of the protein and A FLAGtag to analyze the localization of the protein into the membrane. <br><br />
<li>What: <b>Final check</b><br><br />
Why: check, check, double check!<br><br />
How: Align your final protein sequence to the original sequence of your receptor and the chimeric receptors (can be done by blasting too). Also look in http://tools.neb.com/NEBcutter2/ for forbidden restriction sites.<br><br />
<li><b>Now you can send you sequence to a synthesizing company or work with isolated DNA. </b><br><br />
</ol><br />
<a name="P3"> <br><h2> Example</h2> </a><br/><br />
<br />
For our example we take ORL2156, found on the olfactory database of the university of Yale. <br />
2. The Elm server had the following output on a the protein sequence of ORL2156:<br><br />
<b>Globular domains/ TM domains and signal peptide detected by the SMART server</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/c/c3/Domain.jpg" width="200" align="center"/><br><br />
3. When looking at the cDNA code translated by expasy we indicate the yellow as the cut-off region --> Cut out the yellow parts:<br />
<img src="https://static.igem.org/mediawiki/2012/4/47/Cutoutyellowparts.jpg" width="600"/><br><br />
5. Now the yellow parts are removed both in the cDNA and protein code, the check is not shown.<br />
6. Here the sequence of I7 is added. For convenience we made it a picture.<br><br />
<img src="https://static.igem.org/mediawiki/2012/3/30/Substitute.jpg" width="600"/><br><br />
7. Codon optimization is performed.<br><br />
<img src="https://static.igem.org/mediawiki/2012/f/ff/Optimalization.jpg" width="400"/><br><br />
8. A very important aspect: adding features! This is of course totally dependant on the methods you want to integrate the designed receptor... So we will leave it to your own creativity.<br />
9. The checking:<br />
Protein alignment with original receptor:<br><br />
<img src="https://static.igem.org/mediawiki/2012/2/20/Align.jpg" width="500"/><br><br />
Protein alignment with Rat receptor:<br><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Comparison_RI.jpg" width="500"/><br><br />
Region detection by the elm server:<br><br />
<img src="https://static.igem.org/mediawiki/2012/9/96/Laststep_domain.jpg" width="200" align="center"/><br><br />
Looks good! All 22 amino acids through the membrane. Now we can synthesize the construct or design primer to perform PCR on the species used. Also we can run the YASARA program to optimize the binding niche for the specific ligand and then synthesize!<br><br><br />
<br />
<br />
<a name="P9"><h2>References</h2> </a><br />
<h6>[1] Venkat Radhika, Tassula Proikas-Cezanne, Muralidharan Jayaraman, Djamila Onesime, Ji Hee Ha & Danny N Dhanasekaran, Chemical sensing of DNT by engineered olfactory yeast strain, Nature Chemical biology (2007)<br><br />
[2]Janet M. Young et al. Different evolutionary processes shaped the mouse and human olfactory receptor gene families Hum. Mol. Gen. 2002, Vol. 11, No. 5<br/><br />
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<a href='https://2012.igem.org/Main_Page' target="_blank"><div id='logo_igem2'></div><a/></body></html></div>MarkWeijershttp://2012.igem.org/Team:TU-Delft/receptordesignTeam:TU-Delft/receptordesign2012-10-27T02:46:33Z<p>MarkWeijers: </p>
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<head><title>Receptor</title></head><br />
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<div style="height:70px; width:100%;"></div><br />
<div id="logo_ed"><a href="https://2012.igem.org/Team:TU-Delft" 'onfocus=this.blur()'><img src="https://static.igem.org/mediawiki/igem.org/8/88/Logoigemklein.png" border="0" width="100" height="100"/></a></div><br />
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<img src="https://static.igem.org/mediawiki/igem.org/3/3e/ReceptorDesign.jpg" align="middle" width="100%"/><br />
<br />
<br />
<h2>Content</h2> <br />
<a href="#P4">Chimeric receptor design</a><br><br />
<a href="#P1">DO IT YOURSELF receptor design: What, Why and How</a><br><br />
<a href="#P2">In silico protocol</a><br><br />
<a href="#P3">Example</a><br><br />
<a href="#P9">References</a><br><br />
<br />
<a name="P4"> <br><h2> Chimeric receptor design</h2> </a> <br />
<p>A major hindrance for functional expression of ORs has been that the receptors did not localize in the membrane or that the downstream coupling of the receptor to the Gα did not work properly. It has been shown that the rat olfactory receptor 17 (R17) that responds to octanal can be functionally expressed in many different cell types, including <i>S. cerevisiae</i> [6]. Earlier research investigated on the question whether the RI7 sequence can be used to functionally express other ORs. Sequence analysis of ORs have shown that the N-termini of the receptor are involved in plasma membrane localization, whereas the C-termini generally define the specificity for G protein interaction [7]. Based on this observations <i> Radhika et al.</i> functionally expressed a chimeric OR with the N-terminus and the C-terminus of the RI7 sequence. A schematic picture is shown in figure 2. In this iGEM project we use the same approach as <i>Radhika et al.</i> by substituting the receptor termini with the RI7 sequences.</p><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/3/36/Chimeric_design.png"height="200" width="350" /><br />
<h6>Schematic overview of the chimeric design of the receptor. Figure adapted from <i>Radhika et al.</i>.</h6><br />
<br />
<a name="P1"> <br><h2> DO YOURSELF receptor design: What, Why and How</h2> </a><br />
<br />
<h3>What?</h3> <br />
Protocol for making protein chimeras with a rat G protein coupled receptor (RI7) and Your Favorite Receptor. The order of the DNA sequence looks like this: RI7-[Your Favorite Receptor]-RI7<br />
<h3>Why?</h3><br />
One of the requirements for a working GPCR is that the receptor should be localized into the outside membrane of yeast cell. By replacing the N-terminal part of Your Favorite Receptor by the N-terminal ends of a receptor that is known to be localized into the outside membrane of Saccharomyces cerevisiae (R17), Your Favorite Receptor (YFR) will also be localized into the membrane. The C-terminal part of a GPCR is the alpha subunit binding region. If this is replaced by the RI7 regions a higher affinity with the alpha subunit can be reached [1].<br />
<h3>How?</h3><br />
With this step-by-step protocol we guide you trough all the in silico designing steps. After this the DNA can be transformed in yeast and you have your own olfactory yeast!<br />
<br/><br />
<br />
<br />
<a name="P2"> <br><h2> In silico protocol</h2> </a><br><br />
<ol><li>What: <b>Get your receptor protein sequence code </b><br><br />
Why: To introduce a new receptor chimera in yeast you should start with a GPCR with at least a known sequence and preferably a known ligand.<br><br />
How: By using earlier research on GPCRs. For example a nice GPCR database is http://senselab.med.yale.edu/OdorDB/. Copy the DNA sequence and the protein sequence in a text file. <br><br />
<li>What: <b>look for the transmembrane regions</b><br><br />
Why: Normally GPCRs have seven transmembrane regions. The N-terminal loop is important for the localization in the membrane and should be replaced by the RI7 N-terminal sequence. The C-terminal region directly after the last transmembrane part codes for the alpha-subunit binding region. If this region is replaced by the RI7 region a higher affinity with the alpha subunit can be reached.<br><br />
How: Go to http://elm.eu.org/, enter the protein sequence code and find protein motifs for Saccharomyces cerevisiae and the original species. Compare the Global domain table.<br />
Ideally it finds seven transmembrane regions that all have approximately the same length (quite a conserved domains are found[2] ). <br />
When this is not the case, investigate the hydrophobicity by a hydrophobicity index (analysis can be done by Matlab Bioinformatics tool, but this less conclusive due to multiple hydrophobicity indexes). <br><br />
<li>What: <b>Remove protein sequences that code for the N-terminal and C-terminal regions</b><br><br />
Why: The sequence that code for the N-terminal loop should be replaced by the RI7 sequence for better membrane localization. The C-terminal region should be replaced for a higher affinity with the alpha subunit.<br><br />
How: Delete the sequence at the N-terminal end directly after the first transmembrane part (when read from N=left to C=right). <br><br />
Delete the C terminus directly after the last transmembrane part, this is the subunit binding region.<br><br />
<li>What: <b>Check what you did so far</b><br><br />
Why: You want to know if you removed the right regions. Do I have receptor with only six transmembrane regions? <br><br />
How: Check with http://elm.eu.org/, enter the protein sequence code and find protein motifs.<br><br />
<li>What: <b>Go from protein sequence to DNA sequence</b><br><br />
Why: For further adaptations it is easier to work with the DNA sequence<br><br />
How: Enter the original full length DNA sequence in http://web.expasy.org/translate/ with the output format “Include nucleotide sequence’. Now you can easily find which nucleotides should be removed. <br><br />
<li>What: <b>Add the RI7 N-terminal DNA code upstream of your DNA sequence and the RI7 C-terminal downstream</b><br><br />
Why: The sequence that code for the N-terminal loop should be replaced by the RI7 sequence for better membrane localization. The C-terminal region should be replaced for a higher affinity with the alpha subunit.<br><br />
How: Go to Biobrick BBa_K775000 in the Registry of Standard Biological Parts, copy the RI7 N-terminal parts and paste this upstream of YFR sequence. Copy also the RI7 N-terminal parts and paste this downstream of YFR sequence.<br><br />
<li>What: <b>Codon optimize the sequence for Saccharomyce cerevisiae</b><br><br />
Why: For better expression of the protein in yeast<br><br />
How: Go to http://www.jcat.de/ and enter the sequence. Also specify the restriction sites that you don’t want to have in the sequence: at least the standard illegal restriction sites: EcoRI, XbaI, PsteI, SpeI.<br><br />
<li>What: <b>add BamHI and NdeI restriction sites and other features</b><br><br />
Why: If you have this two restriction sites you can easily clone your receptor in BBa_K775000 to have a yeast promoter and terminator.<br><br />
How: If you send it for synthesizing just add the nucleotides in your file. If you work with cDNA you can add the restriction sites with PCR. <br><br />
Tip: if you send your sequence for synthesizing you can also add a Kozak sequence for better translation of the protein and A FLAGtag to analyze the localization of the protein into the membrane. <br><br />
<li>What: <b>Final check</b><br><br />
Why: check, check, double check!<br><br />
How: Align your final protein sequence to the original sequence of your receptor and the chimeric receptors (can be done by blasting too). Also look in http://tools.neb.com/NEBcutter2/ for forbidden restriction sites.<br><br />
<li><b>Now you can send you sequence to a synthesizing company or work with isolated DNA. </b><br><br />
</ol><br />
<a name="P3"> <br><h2> Example</h2> </a><br/><br />
<br />
For our example we take ORL2156, found on the olfactory database of the university of Yale. <br />
2. The Elm server had the following output on a the protein sequence of ORL2156:<br><br />
<b>Globular domains/ TM domains and signal peptide detected by the SMART server</b><br><br />
<img src="https://static.igem.org/mediawiki/2012/c/c3/Domain.jpg" width="400"/><br><br />
3. When looking at the cDNA code translated by expasy we indicate the yellow as the cut-off region --> Cut out the yellow parts:<br />
<img src="https://static.igem.org/mediawiki/2012/4/47/Cutoutyellowparts.jpg" width="600"/><br><br />
5. Now the yellow parts are removed both in the cDNA and protein code, the check is not shown.<br />
6. Here the sequence of I7 is added. For convenience we made it a picture.<br><br />
<img src="https://static.igem.org/mediawiki/2012/3/30/Substitute.jpg" width="600"/><br><br />
7. Codon optimization is performed.<br><br />
<img src="https://static.igem.org/mediawiki/2012/f/ff/Optimalization.jpg" width="600"/><br><br />
8. A very important aspect: adding features! This is of course totally dependant on the methods you want to integrate the designed receptor... So we will leave it to your own creativity.<br />
9. The checking:<br />
Protein alignment with original receptor:<br><br />
<img src="https://static.igem.org/mediawiki/2012/2/20/Align.jpg" width="500"/><br><br />
Protein alignment with Rat receptor:<br><br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Comparison_RI.jpg" width="500"/><br><br />
Region detection by the elm server:<br><br />
<img src="https://static.igem.org/mediawiki/2012/9/96/Laststep_domain.jpg" width="500"/><br><br />
Looks good! All 22 amino acids through the membrane. Now we can synthesize the construct or design primer to perform PCR on the species used. Also we can run the YASARA program to optimize the binding niche for the specific ligand and then synthesize!<br><br><br />
<br />
<br />
<a name="P9"><h2>References</h2> </a><br />
<h6>[1] Venkat Radhika, Tassula Proikas-Cezanne, Muralidharan Jayaraman, Djamila Onesime, Ji Hee Ha & Danny N Dhanasekaran, Chemical sensing of DNT by engineered olfactory yeast strain, Nature Chemical biology (2007)<br><br />
[2]Janet M. Young et al. Different evolutionary processes shaped the mouse and human olfactory receptor gene families Hum. Mol. Gen. 2002, Vol. 11, No. 5<br/><br />
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<a href='https://2012.igem.org/Main_Page' target="_blank"><div id='logo_igem2'></div><a/></body></html></div>MarkWeijershttp://2012.igem.org/Team:TU-Delft/receptordesignTeam:TU-Delft/receptordesign2012-10-27T02:44:24Z<p>MarkWeijers: </p>
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<br />
<h2>Content</h2> <br />
<a href="#P4">Chimeric receptor design</a><br><br />
<a href="#P1">DO IT YOURSELF receptor design: What, Why and How</a><br><br />
<a href="#P2">In silico protocol</a><br><br />
<a href="#P3">Example</a><br><br />
<a href="#P9">References</a><br><br />
<br />
<a name="P4"> <br><h2> Chimeric receptor design</h2> </a> <br />
<p>A major hindrance for functional expression of ORs has been that the receptors did not localize in the membrane or that the downstream coupling of the receptor to the Gα did not work properly. It has been shown that the rat olfactory receptor 17 (R17) that responds to octanal can be functionally expressed in many different cell types, including <i>S. cerevisiae</i> [6]. Earlier research investigated on the question whether the RI7 sequence can be used to functionally express other ORs. Sequence analysis of ORs have shown that the N-termini of the receptor are involved in plasma membrane localization, whereas the C-termini generally define the specificity for G protein interaction [7]. Based on this observations <i> Radhika et al.</i> functionally expressed a chimeric OR with the N-terminus and the C-terminus of the RI7 sequence. A schematic picture is shown in figure 2. In this iGEM project we use the same approach as <i>Radhika et al.</i> by substituting the receptor termini with the RI7 sequences.</p><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/3/36/Chimeric_design.png"height="200" width="350" /><br />
<h6>Schematic overview of the chimeric design of the receptor. Figure adapted from <i>Radhika et al.</i>.</h6><br />
<br />
<a name="P1"> <br><h2> DO YOURSELF receptor design: What, Why and How</h2> </a><br />
<br />
<h3>What?</h3> <br />
Protocol for making protein chimeras with a rat G protein coupled receptor (RI7) and Your Favorite Receptor. The order of the DNA sequence looks like this: RI7-[Your Favorite Receptor]-RI7<br />
<h3>Why?</h3><br />
One of the requirements for a working GPCR is that the receptor should be localized into the outside membrane of yeast cell. By replacing the N-terminal part of Your Favorite Receptor by the N-terminal ends of a receptor that is known to be localized into the outside membrane of Saccharomyces cerevisiae (R17), Your Favorite Receptor (YFR) will also be localized into the membrane. The C-terminal part of a GPCR is the alpha subunit binding region. If this is replaced by the RI7 regions a higher affinity with the alpha subunit can be reached [1].<br />
<h3>How?</h3><br />
With this step-by-step protocol we guide you trough all the in silico designing steps. After this the DNA can be transformed in yeast and you have your own olfactory yeast!<br />
<br/><br />
<br />
<br />
<a name="P2"> <br><h2> In silico protocol</h2> </a><br><br />
<ol><li>What: <b>Get your receptor protein sequence code </b><br><br />
Why: To introduce a new receptor chimera in yeast you should start with a GPCR with at least a known sequence and preferably a known ligand.<br><br />
How: By using earlier research on GPCRs. For example a nice GPCR database is http://senselab.med.yale.edu/OdorDB/. Copy the DNA sequence and the protein sequence in a text file. <br><br />
<li>What: <b>look for the transmembrane regions</b><br><br />
Why: Normally GPCRs have seven transmembrane regions. The N-terminal loop is important for the localization in the membrane and should be replaced by the RI7 N-terminal sequence. The C-terminal region directly after the last transmembrane part codes for the alpha-subunit binding region. If this region is replaced by the RI7 region a higher affinity with the alpha subunit can be reached.<br><br />
How: Go to http://elm.eu.org/, enter the protein sequence code and find protein motifs for Saccharomyces cerevisiae and the original species. Compare the Global domain table.<br />
Ideally it finds seven transmembrane regions that all have approximately the same length (quite a conserved domains are found[2] ). <br />
When this is not the case, investigate the hydrophobicity by a hydrophobicity index (analysis can be done by Matlab Bioinformatics tool, but this less conclusive due to multiple hydrophobicity indexes). <br><br />
<li>What: <b>Remove protein sequences that code for the N-terminal and C-terminal regions</b><br><br />
Why: The sequence that code for the N-terminal loop should be replaced by the RI7 sequence for better membrane localization. The C-terminal region should be replaced for a higher affinity with the alpha subunit.<br><br />
How: Delete the sequence at the N-terminal end directly after the first transmembrane part (when read from N=left to C=right). <br><br />
Delete the C terminus directly after the last transmembrane part, this is the subunit binding region.<br><br />
<li>What: <b>Check what you did so far</b><br><br />
Why: You want to know if you removed the right regions. Do I have receptor with only six transmembrane regions? <br><br />
How: Check with http://elm.eu.org/, enter the protein sequence code and find protein motifs.<br><br />
<li>What: <b>Go from protein sequence to DNA sequence</b><br><br />
Why: For further adaptations it is easier to work with the DNA sequence<br><br />
How: Enter the original full length DNA sequence in http://web.expasy.org/translate/ with the output format “Include nucleotide sequence’. Now you can easily find which nucleotides should be removed. <br><br />
<li>What: <b>Add the RI7 N-terminal DNA code upstream of your DNA sequence and the RI7 C-terminal downstream</b><br><br />
Why: The sequence that code for the N-terminal loop should be replaced by the RI7 sequence for better membrane localization. The C-terminal region should be replaced for a higher affinity with the alpha subunit.<br><br />
How: Go to Biobrick BBa_K775000 in the Registry of Standard Biological Parts, copy the RI7 N-terminal parts and paste this upstream of YFR sequence. Copy also the RI7 N-terminal parts and paste this downstream of YFR sequence.<br><br />
<li>What: <b>Codon optimize the sequence for Saccharomyce cerevisiae</b><br><br />
Why: For better expression of the protein in yeast<br><br />
How: Go to http://www.jcat.de/ and enter the sequence. Also specify the restriction sites that you don’t want to have in the sequence: at least the standard illegal restriction sites: EcoRI, XbaI, PsteI, SpeI.<br><br />
<li>What: <b>add BamHI and NdeI restriction sites and other features</b><br><br />
Why: If you have this two restriction sites you can easily clone your receptor in BBa_K775000 to have a yeast promoter and terminator.<br><br />
How: If you send it for synthesizing just add the nucleotides in your file. If you work with cDNA you can add the restriction sites with PCR. <br><br />
Tip: if you send your sequence for synthesizing you can also add a Kozak sequence for better translation of the protein and A FLAGtag to analyze the localization of the protein into the membrane. <br><br />
<li>What: <b>Final check</b><br><br />
Why: check, check, double check!<br><br />
How: Align your final protein sequence to the original sequence of your receptor and the chimeric receptors (can be done by blasting too). Also look in http://tools.neb.com/NEBcutter2/ for forbidden restriction sites.<br><br />
<li><b>Now you can send you sequence to a synthesizing company or work with isolated DNA. </b><br><br />
</ol><br />
<a name="P3"> <br><h2> Example</h2> </a><br/><br />
<br />
For our example we take ORL2156, found on the olfactory database of the university of Yale. <br />
2. The Elm server had the following output on a the protein sequence of ORL2156:<br />
<b>Globular domains/ TM domains and signal peptide detected by the SMART server</b><br />
<img src="https://static.igem.org/mediawiki/2012/c/c3/Domain.jpg" width="200"/><br><br />
3. When looking at the cDNA code translated by expasy we indicate the yellow as the cut-off region --> Cut out the yellow parts:<br />
<img src="https://static.igem.org/mediawiki/2012/4/47/Cutoutyellowparts.jpg" width="200"/><br><br />
5. Now the yellow parts are removed both in the cDNA and protein code, the check is not shown.<br />
6. Here the sequence of I7 is added. For convenience we made it a picture.<br />
<img src="https://static.igem.org/mediawiki/2012/3/30/Substitute.jpg" width="200"/><br><br />
7. Codon optimization is performed.<br />
<img src="https://static.igem.org/mediawiki/2012/f/ff/Optimalization.jpg" width="200"/><br><br />
8. A very important aspect: adding features! This is of course totally dependant on the methods you want to integrate the designed receptor... So we will leave it to your own creativity.<br />
9. The checking:<br />
Protein alignment with original receptor:<br />
<img src="https://static.igem.org/mediawiki/2012/2/20/Align.jpg" width="200"/><br><br />
Protein alignment with Rat receptor:<br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Comparison_RI.jpg" width="200"/><br><br />
Region detection by the elm server:<br />
<img src="https://static.igem.org/mediawiki/2012/9/96/Laststep_domain.jpg" width="200"/><br><br />
Looks good! All 22 amino acids through the membrane. Now we can synthesize the construct or design primer to perform PCR on the species used. Also we can run the YASARA program to optimize the binding niche for the specific ligand and then synthesize!<br><br><br />
<br />
<br />
<a name="P9"><h2>References</h2> </a><br />
<h6>[1] Venkat Radhika, Tassula Proikas-Cezanne, Muralidharan Jayaraman, Djamila Onesime, Ji Hee Ha & Danny N Dhanasekaran, Chemical sensing of DNT by engineered olfactory yeast strain, Nature Chemical biology (2007)<br><br />
[2]Janet M. Young et al. Different evolutionary processes shaped the mouse and human olfactory receptor gene families Hum. Mol. Gen. 2002, Vol. 11, No. 5<br/><br />
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<a href='https://2012.igem.org/Main_Page' target="_blank"><div id='logo_igem2'></div><a/></body></html></div>MarkWeijershttp://2012.igem.org/Team:TU-Delft/receptordesignTeam:TU-Delft/receptordesign2012-10-27T02:42:16Z<p>MarkWeijers: </p>
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<head><title>Receptor</title></head><br />
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<div id="logo_ed"><a href="https://2012.igem.org/Team:TU-Delft" 'onfocus=this.blur()'><img src="https://static.igem.org/mediawiki/igem.org/8/88/Logoigemklein.png" border="0" width="100" height="100"/></a></div><br />
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<br />
<br />
<h2>Content</h2> <br />
<a href="#P4">Chimeric receptor design</a><br><br />
<a href="#P1">DO IT YOURSELF receptor design: What, Why and How</a><br><br />
<a href="#P2">In silico protocol</a><br><br />
<a href="#P3">Example</a><br><br />
<a href="#P9">References</a><br><br />
<br />
<a name="P4"> <br><h2> Chimeric receptor design</h2> </a> <br />
<p>A major hindrance for functional expression of ORs has been that the receptors did not localize in the membrane or that the downstream coupling of the receptor to the Gα did not work properly. It has been shown that the rat olfactory receptor 17 (R17) that responds to octanal can be functionally expressed in many different cell types, including <i>S. cerevisiae</i> [6]. Earlier research investigated on the question whether the RI7 sequence can be used to functionally express other ORs. Sequence analysis of ORs have shown that the N-termini of the receptor are involved in plasma membrane localization, whereas the C-termini generally define the specificity for G protein interaction [7]. Based on this observations <i> Radhika et al.</i> functionally expressed a chimeric OR with the N-terminus and the C-terminus of the RI7 sequence. A schematic picture is shown in figure 2. In this iGEM project we use the same approach as <i>Radhika et al.</i> by substituting the receptor termini with the RI7 sequences.</p><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/3/36/Chimeric_design.png"height="200" width="350" /><br />
<h6>Schematic overview of the chimeric design of the receptor. Figure adapted from <i>Radhika et al.</i>.</h6><br />
<br />
<a name="P1"> <br><h2> DO YOURSELF receptor design: What, Why and How</h2> </a><br />
<br />
<h3>What?</h3> <br />
Protocol for making protein chimeras with a rat G protein coupled receptor (RI7) and Your Favorite Receptor. The order of the DNA sequence looks like this: RI7-[Your Favorite Receptor]-RI7<br />
<h3>Why?</h3><br />
One of the requirements for a working GPCR is that the receptor should be localized into the outside membrane of yeast cell. By replacing the N-terminal part of Your Favorite Receptor by the N-terminal ends of a receptor that is known to be localized into the outside membrane of Saccharomyces cerevisiae (R17), Your Favorite Receptor (YFR) will also be localized into the membrane. The C-terminal part of a GPCR is the alpha subunit binding region. If this is replaced by the RI7 regions a higher affinity with the alpha subunit can be reached [1].<br />
<h3>How?</h3><br />
With this step-by-step protocol we guide you trough all the in silico designing steps. After this the DNA can be transformed in yeast and you have your own olfactory yeast!<br />
<br/><br />
<br />
<br />
<a name="P2"> <br><h2> In silico protocol</h2> </a><br><br />
<ol><li>What: <b>Get your receptor protein sequence code </b><br><br />
Why: To introduce a new receptor chimera in yeast you should start with a GPCR with at least a known sequence and preferably a known ligand.<br><br />
How: By using earlier research on GPCRs. For example a nice GPCR database is http://senselab.med.yale.edu/OdorDB/. Copy the DNA sequence and the protein sequence in a text file. <br><br />
<li>What: <b>look for the transmembrane regions</b><br><br />
Why: Normally GPCRs have seven transmembrane regions. The N-terminal loop is important for the localization in the membrane and should be replaced by the RI7 N-terminal sequence. The C-terminal region directly after the last transmembrane part codes for the alpha-subunit binding region. If this region is replaced by the RI7 region a higher affinity with the alpha subunit can be reached.<br><br />
How: Go to http://elm.eu.org/, enter the protein sequence code and find protein motifs for Saccharomyces cerevisiae and the original species. Compare the Global domain table.<br />
Ideally it finds seven transmembrane regions that all have approximately the same length (quite a conserved domains are found[2] ). <br />
When this is not the case, investigate the hydrophobicity by a hydrophobicity index (analysis can be done by Matlab Bioinformatics tool, but this less conclusive due to multiple hydrophobicity indexes). <br><br />
<li>What: <b>Remove protein sequences that code for the N-terminal and C-terminal regions</b><br><br />
Why: The sequence that code for the N-terminal loop should be replaced by the RI7 sequence for better membrane localization. The C-terminal region should be replaced for a higher affinity with the alpha subunit.<br><br />
How: Delete the sequence at the N-terminal end directly after the first transmembrane part (when read from N=left to C=right). <br><br />
Delete the C terminus directly after the last transmembrane part, this is the subunit binding region.<br><br />
<li>What: <b>Check what you did so far</b><br><br />
Why: You want to know if you removed the right regions. Do I have receptor with only six transmembrane regions? <br><br />
How: Check with http://elm.eu.org/, enter the protein sequence code and find protein motifs.<br><br />
<li>What: <b>Go from protein sequence to DNA sequence</b><br><br />
Why: For further adaptations it is easier to work with the DNA sequence<br><br />
How: Enter the original full length DNA sequence in http://web.expasy.org/translate/ with the output format “Include nucleotide sequence’. Now you can easily find which nucleotides should be removed. <br><br />
<li>What: <b>Add the RI7 N-terminal DNA code upstream of your DNA sequence and the RI7 C-terminal downstream</b><br><br />
Why: The sequence that code for the N-terminal loop should be replaced by the RI7 sequence for better membrane localization. The C-terminal region should be replaced for a higher affinity with the alpha subunit.<br><br />
How: Go to Biobrick BBa_K775000 in the Registry of Standard Biological Parts, copy the RI7 N-terminal parts and paste this upstream of YFR sequence. Copy also the RI7 N-terminal parts and paste this downstream of YFR sequence.<br><br />
<li>What: <b>Codon optimize the sequence for Saccharomyce cerevisiae</b><br><br />
Why: For better expression of the protein in yeast<br><br />
How: Go to http://www.jcat.de/ and enter the sequence. Also specify the restriction sites that you don’t want to have in the sequence: at least the standard illegal restriction sites: EcoRI, XbaI, PsteI, SpeI.<br><br />
<li>What: <b>add BamHI and NdeI restriction sites and other features</b><br><br />
Why: If you have this two restriction sites you can easily clone your receptor in BBa_K775000 to have a yeast promoter and terminator.<br><br />
How: If you send it for synthesizing just add the nucleotides in your file. If you work with cDNA you can add the restriction sites with PCR. <br><br />
Tip: if you send your sequence for synthesizing you can also add a Kozak sequence for better translation of the protein and A FLAGtag to analyze the localization of the protein into the membrane. <br><br />
<li>What: <b>Final check</b><br><br />
Why: check, check, double check!<br><br />
How: Align your final protein sequence to the original sequence of your receptor and the chimeric receptors (can be done by blasting too). Also look in http://tools.neb.com/NEBcutter2/ for forbidden restriction sites.<br><br />
<li><b>Now you can send you sequence to a synthesizing company or work with isolated DNA. </b><br><br />
</ol><br />
<a name="P3"> <br><h2> Example</h2> </a><br/><br />
<br />
For our example we take ORL2156, found on the olfactory database of the university of Yale. <br />
2. The Elm server had the following output on a the protein sequence of ORL2156:<br />
<b>Globular domains/ TM domains and signal peptide detected by the SMART server</b><br />
<img src="https://static.igem.org/mediawiki/2012/c/c3/Domain.jpg"/><br><br />
3. When looking at the cDNA code translated by expasy we indicate the yellow as the cut-off region --> Cut out the yellow parts:<br />
<img src="https://static.igem.org/mediawiki/2012/4/47/Cutoutyellowparts.jpg"/><br><br />
5. Now the yellow parts are removed both in the cDNA and protein code, the check is not shown.<br />
6. Here the sequence of I7 is added. For convenience we made it a picture.<br />
<img src="https://static.igem.org/mediawiki/2012/3/30/Substitute.jpg"/><br><br />
7. Codon optimization is performed.<br />
<img src="https://static.igem.org/mediawiki/2012/f/ff/Optimalization.jpg"/><br><br />
8. A very important aspect: adding features! This is of course totally dependant on the methods you want to integrate the designed receptor... So we will leave it to your own creativity.<br />
9. The checking:<br />
Protein alignment with original receptor:<br />
<img src="https://static.igem.org/mediawiki/2012/2/20/Align.jpg"/><br><br />
Protein alignment with Rat receptor:<br />
<img src="https://static.igem.org/mediawiki/2012/8/85/Comparison_RI.jpg"/><br><br />
Region detection by the elm server:<br />
<img src="https://static.igem.org/mediawiki/2012/9/96/Laststep_domain.jpg"/><br><br />
Looks good! All 22 amino acids through the membrane. Now we can synthesize the construct or design primer to perform PCR on the species used. Also we can run the YASARA program to optimize the binding niche for the specific ligand and then synthesize!<br><br><br />
<br />
<br />
<a name="P9"><h2>References</h2> </a><br />
<h6>[1] Venkat Radhika, Tassula Proikas-Cezanne, Muralidharan Jayaraman, Djamila Onesime, Ji Hee Ha & Danny N Dhanasekaran, Chemical sensing of DNT by engineered olfactory yeast strain, Nature Chemical biology (2007)<br><br />
[2]Janet M. Young et al. Different evolutionary processes shaped the mouse and human olfactory receptor gene families Hum. Mol. Gen. 2002, Vol. 11, No. 5<br/><br />
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<a href='https://2012.igem.org/Main_Page' target="_blank"><div id='logo_igem2'></div><a/></body></html></div>MarkWeijershttp://2012.igem.org/File:Substitute.jpgFile:Substitute.jpg2012-10-27T02:25:09Z<p>MarkWeijers: </p>
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<div></div>MarkWeijershttp://2012.igem.org/File:Optimalization.jpgFile:Optimalization.jpg2012-10-27T02:24:50Z<p>MarkWeijers: </p>
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<div></div>MarkWeijershttp://2012.igem.org/File:Laststep_domain.jpgFile:Laststep domain.jpg2012-10-27T02:24:30Z<p>MarkWeijers: </p>
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<div></div>MarkWeijershttp://2012.igem.org/File:Domain.jpgFile:Domain.jpg2012-10-27T02:24:10Z<p>MarkWeijers: </p>
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<div></div>MarkWeijershttp://2012.igem.org/File:Cutoutyellowparts.jpgFile:Cutoutyellowparts.jpg2012-10-27T02:23:54Z<p>MarkWeijers: </p>
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<div></div>MarkWeijershttp://2012.igem.org/File:Comparison_RI.jpgFile:Comparison RI.jpg2012-10-27T02:23:31Z<p>MarkWeijers: </p>
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<div></div>MarkWeijershttp://2012.igem.org/File:Align.jpgFile:Align.jpg2012-10-27T02:22:17Z<p>MarkWeijers: </p>
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<div></div>MarkWeijershttp://2012.igem.org/Team:TU-Delft/HPTeam:TU-Delft/HP2012-10-27T01:07:57Z<p>MarkWeijers: </p>
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<head><title>Human Practice</title><br />
<body><br />
<div style="height:70px; width:100%;"></div> <br />
<img src="https://static.igem.org/mediawiki/igem.org/7/74/Background_greenlamp.jpg" class="bg_team"><br />
<div id="logo_ed"><a href="https://2012.igem.org/Team:TU-Delft" 'onfocus=this.blur()'><img src="https://static.igem.org/mediawiki/igem.org/8/88/Logoigemklein.png" border="0" width="100" height="100"></a></div><br />
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<h2>Snifferomyces - A solution for world problems?</h2><br><br />
<p>This is a very complicated question. When we started the project, first was determined on which possible solutions we will perform study:<br />
<ul><br />
<li>Tuberculosis Detection<br />
<li>Detection of explosives<br />
</ul><br />
The tuberculosis detection was our main case study and a lot of wetlab work on this possible methyl nicotinate receptor has been done.<br />
The detection of explosives is a application we thought of was inspired by the K-9's detecting explosives. Reportings of sensing DNT by yeast strains (Venkat Radhika et al. (2007)) supported this.<br />
<br/><br />
<h4>Is Snifferomyces the solution for tuberculosis detection?</h4><br />
This question is approached on two different ways. A interview with Dr. Rene Lutter and a implementation study. For the implementation study we subdivided<br />
this question into several smaller, more specific questions.<br />
<ul><br />
<li>What is tuberculosis?<br />
<li>What are the present diagnostic tools?<br />
<li>Why would a diagnostic tool based on our principle contribute?<br />
<li>What does the problem area look like?<br />
<li>What would be the possible drawbacks during R&D and usage?<br />
<br />
</ul><br />
<h4>Is Snifferomyces the solution for explosive detection?</h4><br />
In this case we didn't do a wetlab study but spoke to the <i>Royal Dutch Military Police</i>. The Royal Dutch Military Police are present<br />
at Schiphol to detect possible threats to the National Security. One part of their job is to be on the lookout for explosives. This is mainly<br />
done by K-9's, we asked their opinion about using micro organisms and GMO's as explosive detectors!<br />
</p><br />
<center><h3>Please click on the photo's to see the project</h3></center><br />
<center><br />
<table id="tbtext"><br />
<tr><br />
<th>Military Police</th><br />
<th>Implementation Study</th><br />
<th>Interview with Doctor</th><br />
</tr><br />
<tr><br />
<td><a href="https://2012.igem.org/Team:TU-Delft/HP/MP" target="_blank"> <br />
<img src="https://static.igem.org/mediawiki/igem.org/9/9a/Hond.jpg" height="130" width="189"/></a></td><br />
<td><a href="https://2012.igem.org/Team:TU-Delft/HP/Study" target="_blank"> <br />
<img src="https://static.igem.org/mediawiki/igem.org/b/b2/HospitalILHAM.JPG" height="130" width="189"/></a></td><br />
<td><a href="https://2012.igem.org/Team:TU-Delft/HP/DocTalk" target="_blank"> <br />
<img src="https://static.igem.org/mediawiki/igem.org/d/d2/Stethoscope-2.png" height="130" width="189"/></a></td><br />
</tr></table><br />
</center><br />
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<a href='https://2012.igem.org/Main_Page' target="_blank"><div id='logo_igem2'></div><a/> </body></html></div>MarkWeijershttp://2012.igem.org/Team:TU-Delft/HPTeam:TU-Delft/HP2012-10-27T01:01:20Z<p>MarkWeijers: </p>
<hr />
<div>{{:Team:TUDelft/Scripts}}{{:Team:TUDelft/CSS}}{{:Team:TUDelft/menu}}<br />
<html><br />
<head><title>Human Practice</title><br />
<body><br />
<div style="height:70px; width:100%;"></div> <br />
<img src="https://static.igem.org/mediawiki/igem.org/7/74/Background_greenlamp.jpg" class="bg_team"><br />
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<img src="https://static.igem.org/mediawiki/igem.org/e/ed/Nieuwe_header_human_practices.jpg" align="middle" width="100%"><br />
<br/><br />
<br/><br />
<br />
<h2>Snifferomyces - A solution for world problems?</h2><br><br />
<p>This is a very complicated question. When we started the project, first was determined on which possible solutions we will perform study:<br />
<ul><br />
<li>Tuberculosis Detection<br />
<li>Detection of explosives<br />
</ul><br />
The tuberculosis detection was our main case study and a lot of wetlab work on this possible methyl nicotinate receptor has been done.<br />
The detection of explosives is a application we thought of was inspired by the K-9's detecting explosives. Reportings of sensing DNT by yeast strains (Venkat Radhika et al. (2007)) supported this.<br />
<br/><br />
<h4>Is Snifferomyces the solution for tuberculosis detection?</h4><br />
This question is approached on two different ways. A interview with Dr. Rene Lutter and a implementation study. For the implementation study we subdivided<br />
this question into several smaller, more specific questions.<br />
<ul><br />
<li>What is tuberculosis?<br />
<li>What are the present diagnostic tools?<br />
<li>Why would a diagnostic tool based on our principle contribute?<br />
<li>What does the problem area look like?<br />
</ul><br />
<h4>Is Snifferomyces the solution for explosive detection?</h4><br />
In this case we didn't do a wetlab study but spoke to the <i>Royal Dutch Military Police</i>. The Royal Dutch Military Police are present<br />
at Schiphol to detect possible threats to the National Security. One part of their job is to be on the lookout for explosives. This is mainly<br />
done by K-9's, we asked their opinion about using micro organisms and GMO's as explosive detectors!<br />
</p><br />
<center><h3>Please click on the photo's to see the project</h3></center><br />
<center><br />
<table id="tbtext"><br />
<tr><br />
<th>Military Police</th><br />
<th>Implementation Study</th><br />
<th>Interview with Doctor</th><br />
</tr><br />
<tr><br />
<td><a href="https://2012.igem.org/Team:TU-Delft/HP/MP" target="_blank"> <br />
<img src="https://static.igem.org/mediawiki/igem.org/9/9a/Hond.jpg" height="130" width="189"/></a></td><br />
<td><a href="https://2012.igem.org/Team:TU-Delft/HP/Study" target="_blank"> <br />
<img src="https://static.igem.org/mediawiki/igem.org/b/b2/HospitalILHAM.JPG" height="130" width="189"/></a></td><br />
<td><a href="https://2012.igem.org/Team:TU-Delft/HP/DocTalk" target="_blank"> <br />
<img src="https://static.igem.org/mediawiki/igem.org/d/d2/Stethoscope-2.png" height="130" width="189"/></a></td><br />
</tr></table><br />
</center><br />
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<a href='https://2012.igem.org/Main_Page' target="_blank"><div id='logo_igem2'></div><a/> </body></html></div>MarkWeijershttp://2012.igem.org/Team:TU-Delft/SnifferometerTeam:TU-Delft/Snifferometer2012-10-27T00:50:44Z<p>MarkWeijers: </p>
<hr />
<div>{{:Team:TUDelft/Scripts}}{{:Team:TUDelft/CSSLaksh}}{{:Team:TUDelft/menu}}<br />
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<div id="contentbox" style="text-align:justify;"><br />
<br />
In order to assess the feasibility using our Snifferomyces cells as a diagnosis method, a device called "Snifferometer" was built to measure the status of fluorescence. The purpose of building such a device is to overcome the complexity, incalculability, and the high cost of using fluorescence microscope.<br />
= Construction of Snifferometer =<br />
<br />
== Scheme of the Prototype ==<br />
Figure 1 shows the concept of the mechanism of the device. <br />
<br />
The liquid is the mixture of sputum and our snifferomyces. If there are some Tuberculosis smelling cells in sputum, the snifferomyces will respond and produce fluorophores.<br />
<br />
The blue LEDs are to excite the output fluorophores in the snifferomyces yeast. Then the photodiode collects the emitted fluorescence from the yeast and convert it into voltage signal through the amplifying circuit. Thus, by reading the number of voltmeter, the status of TB smelling cells in sputum can be detected.<br />
<br />
With respect to the indicator, because at the moment sufficient data from snifferomyces is absent, we have little of what the voltage value will be with respect to certain amount of sputum and certain amount of snifferomyces. A voltmeter is used as an indicator of the prototype, ideally we indicate the signal with a LED of which the threshold voltage is chosen to be between those in the case of the existence and inexistence of TB cells. When there is Tuberculosis, then light on; otherwise, light off.<br />
<br />
[[File:Scheme of snifferometer.png|270px|left|thumb|'''Figure 1''': Scheme of Snifferometer]]<br />
[[File:snifferometer device.png|250px|right|thumb|'''Figure 2''': Real prototype of Snifferometer]]<br />
<br />
</div><br />
<br />
<div id="contentbox" style="text-align:justify;"><br />
<br />
== Circuit of Snifferometer ==<br />
The photodiode converts the light intensity into small current, and an operational amplifier (op amp) is needed to amplify the small current signal.<br />
<br />
[https://2012.igem.org/File:Component_sheet.PNG Click] to see the component sheet, in which the price is also included. In the sheet, the cost is around 30 euros. In the case of volume production, we can make the price as little as 4 to 10 euros!<br />
[[File:Circuit of Snifferometer.png|580px|Right|thumb|'''Figure 3''': Circuit of Snifferometer]]<br />
</div><br />
<br />
<div id="contentbox" style="text-align:justify;"><br />
<br />
== Wavelength ==<br />
In Snifferomyces, we use EGFP as the output. The [http://flowcyt.salk.edu/fluo.html Table of Fluorochrome] give the peak excitement and emission wavelengthes of EGFP, which are 488nm and 507nm respectively. <br />
<br />
LEDs were chosen to emit light that would excite the EGFP efficiently, thus [http://media.digikey.com/pdf/Data%20Sheets/Optek%20PDFs/OVLFB3C7.pdf Blue LEDs] are chosen with the dominant wavelength of 470nm.<br />
<br />
The photodiode is working as a detector. Because we only care about the existence of emission light with less disturbance of excitement light, a photodiode was chosen that would detect the output with less overlap of the LED spectrum. And the responsivity spectrum of the chosen photodiode can be seen [http://www.marktechopto.com/products/datasheet/MTD5052W here]. <br />
<br />
However, it can be seen that there is still wavelength overlapping from 450 to 500nm between LED spectrum and photodiode spectrum, and photodiode basically works as detector with low accuracy in wavelength filtering. Thus a external high-pass filter is needed which has a clear cutoff frequency around 500nm. In the test, two filters were used to give a comparison:<br />
<br />
<br />
<ul><br />
<li> Filter 1: [http://www.leefilters.com/lighting/colour-details.html#768 LEE Filters 768 Egg yellow].<br />
<li> Filter 2: [http://www.leefilters.com/lighting/colour-details.html#101 LEE Filters 101 yellow].<br />
</ul><br />
<br />
</div><br />
<br />
<div id="contentbox" style="text-align:justify;"><br />
<br />
= Test =<br />
Since the fluorescence signal emitted from yeast is not very strong to reach the sensitivity of our prototype, the ''E.coli'' with TagGFP2 overexpression is then used to test the feasibility of our device. TagGFP2 has 483nm and 506nm for its excitement and emission peak wavelength which is similar with EGFP.<br />
<br />
Eppendorf tubes are used to contain the liquid. The Control sets are M9 medium with ''E.coli'', while the Test sets are M9 medium with ''E.coli'' overexpressing TagGFP2. M9 medium is used because it has low autofluorescence.<br />
<br />
[[File:Msnifferometer.jpg|420px|center|'''Figure4''': Measuring data]]<br />
<br />
<br />
[[File:Data Sniff.png|580px|middle|thumb|'''Table 1''': Measured data]]<br />
<br />
<br />
'''The data of Test sets are higher than these of Control sets, which means Snifferometer is capable to detect the glowing GFP'''. Moreover, the data also shows Filter 2 is better than Filter 1, since its passing high wavelength is overlapping more with the responsivity spectra of photodiode. <br />
<br />
'''In conclusion, the idea of building a Snifferometer is feasible'''. However, there is still room for improvement because the sensitivity of the device is not so satisfactory. Some suggested ways include:<br />
<ul><br />
<li>Reduce light intensity of background. For example, the material of Snifferometer cap can be made with black coarse surface instead of smooth blue, so the reflection of background can be reduced.<br />
<li>Increase light intensity in the yeast cells by pelleting or amplification of the EGFP signal<br />
<li>Enhance the sensitivity of the circuit, for example by increasing the feedback resistor by an op amp. <br />
<li>Enlarge the area of fluorescent: in the test, limited by suitable container, Eppendorf tubes were used, which has a small area to reflect and glow light. We can enhance the received signal for example by using petri dish or plate. <br />
</ul><br />
<html><br />
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<img src="https://static.igem.org/mediawiki/igem.org/3/37/Footer_2.jpg" align="middle" width="690"><br />
<a href='https://2012.igem.org/Main_Page' target="_blank"><div id='logo_igem2'></div><a/></body></html></div>MarkWeijershttp://2012.igem.org/Team:TU-Delft/SnifferometerTeam:TU-Delft/Snifferometer2012-10-27T00:40:26Z<p>MarkWeijers: </p>
<hr />
<div>{{:Team:TUDelft/Scripts}}{{:Team:TUDelft/CSSLaksh}}{{:Team:TUDelft/menu}}<br />
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<div id="contentbox" style="text-align:justify;"><br />
<img src="https://static.igem.org/mediawiki/igem.org/3/30/Sniffer-O-Meter.jpg" align="middle" width="100%"><br />
<br />
</div><br />
</html><br />
<div id="contentbox" style="text-align:justify;"><br />
<br />
In order to assess the feasibility using our Snifferomyces cells as a diagnosis method, a device called "Snifferometer" was built to measure the status of fluorescence. The purpose of building such a device is to overcome the complexity, incalculability, and the high cost of using fluorescence microscope.<br />
= Construction of Snifferometer =<br />
<br />
== Scheme of the Prototype ==<br />
Figure 1 shows the concept of the mechanism of the device. <br />
<br />
The liquid is the mixture of sputum and our snifferomyces. If there are some Tuberculosis smelling cells in sputum, the snifferomyces will respond and produce fluorophores.<br />
<br />
The blue LEDs are to excite the output fluorophores in the snifferomyces yeast. Then the photodiode collects the emitted fluorescence from the yeast and convert it into voltage signal through the amplifying circuit. Thus, by reading the number of voltmeter, the status of TB smelling cells in sputum can be detected.<br />
<br />
With respect to the indicator, because at the moment sufficient data from snifferomyces is absent, we have little of what the voltage value will be with respect to certain amount of sputum and certain amount of snifferomyces. A voltmeter is used as an indicator of the prototype, ideally we indicate the signal with a LED of which the threshold voltage is chosen to be between those in the case of the existence and inexistence of TB cells. When there is Tuberculosis, then light on; otherwise, light off.<br />
<br />
[[File:Scheme of snifferometer.png|270px|left|thumb|'''Figure 1''': Scheme of Snifferometer]]<br />
[[File:snifferometer device.png|250px|right|thumb|'''Figure 2''': Real prototype of Snifferometer]]<br />
<br />
</div><br />
<br />
<div id="contentbox" style="text-align:justify;"><br />
<br />
== Circuit of Snifferometer ==<br />
The photodiode converts the light intensity into small current, and op amp is needed to amplify the small current signal.<br />
<br />
[https://2012.igem.org/File:Component_sheet.PNG Click] to see the component sheet, in which the price is also included. In the sheet, the cost is around 30 euros in the case of volume production. We can make the price as little as 4 to 10 euros if considering the possible lowest price.<br />
[[File:Circuit of Snifferometer.png|580px|Right|thumb|'''Figure 3''': Circuit of Snifferometer]]<br />
</div><br />
<br />
<div id="contentbox" style="text-align:justify;"><br />
<br />
== Wavelength ==<br />
In Snifferomyces, we use EGFP as the output. By looking up in the [http://flowcyt.salk.edu/fluo.html Table of Fluorochrome], the peak excitement and emission wavelengthes of EGFP are known, which are 488nm and 507nm respectively. <br />
<br />
LEDs were chosen to emit light that would excite the EGFP efficiently, thus [http://media.digikey.com/pdf/Data%20Sheets/Optek%20PDFs/OVLFB3C7.pdf Blue LEDs] are chosen with the dominant wavelength of 470nm.<br />
<br />
The photodiode is working as a detector. Because we only care about the existence of emission light with less disturbance of excitement light. A photodiode was chosen that would detect the output with less overlap of the LED spectrum. And the responsivity spectrum of the chosen photodiode can be seen [http://www.marktechopto.com/products/datasheet/MTD5052W here]. <br />
<br />
However, it can be seen that there is still wavelength overlapping from 450 to 500nm between LED spectrum and photodiode spectrum, and photodiode basically works as detector with low accuracy in wavelength filtering. Thus a external high-pass filter is needed which has a clear cutoff frequency around 500nm. In the test, two filters were used to give a comparison:<br />
<br />
<br />
<ul><br />
<li> Filter 1: [http://www.leefilters.com/lighting/colour-details.html#768 LEE Filters 768 Egg yellow].<br />
<li> Filter 2: [http://www.leefilters.com/lighting/colour-details.html#101 LEE Filters 101 yellow].<br />
</ul><br />
<br />
</div><br />
<br />
<div id="contentbox" style="text-align:justify;"><br />
<br />
= Test =<br />
Since the fluorescence signal emitted from yeast is not very strong to reach the sensitivity of our prototype, the ''E.coli'' with TagGFP2 overexpression is then used to test the feasibility of our device. TagGFP2 has 483nm and 506nm for its excitement and emission peak wavelength which is similar with EGFP.<br />
<br />
Eppendorf tubes are used to contain the liquid. The Control sets are M9 medium with ''E.coli'', while the Test sets are M9 medium with ''E.coli'' overexpressing TagGFP2. M9 medium is used because it has low autofluorescence.<br />
<br />
[[File:Msnifferometer.jpg|420px|center|'''Figure4''': Measuring data]]<br />
<br />
<br />
[[File:Data Sniff.png|580px|middle|thumb|'''Table 1''': Measured data]]<br />
<br />
<br />
'''The data of Test sets are higher than these of Control sets, which means Snifferometer is capable to detect the glowing GFP'''. Moreover, the data also shows Filter 2 is better than Filter 1, since its passing high wavelength is overlapping more with the responsivity spectra of photodiode. <br />
<br />
'''In conclusion, the idea of building a Snifferometer is feasible'''. However, there is still room for improvement because the sensitivity of the device is not so satisfactory. Some suggested ways include:<br />
<ul><br />
<li>Reduce light intensity of background. For example, the material of Snifferometer cap can be made with black coarse surface instead of smooth blue, so the reflection of background can be reduced.<br />
<li>Increase green light intensity<br />
<li>Enhance the sensitivity of the circuit, such as increase the feedback resistor of op amp. <br />
<li>Enlarge the area of fluorescent: in the test, limited by suitable container, Eppendorf tubes were used, which has a small area to reflect and glow light. We can enhance the received signal for example by using petri dish or plate. <br />
</ul><br />
<html><br />
<br />
</div><br />
<img src="https://static.igem.org/mediawiki/igem.org/3/37/Footer_2.jpg" align="middle" width="690"><br />
<a href='https://2012.igem.org/Main_Page' target="_blank"><div id='logo_igem2'></div><a/></body></html></div>MarkWeijershttp://2012.igem.org/Team:TU-Delft/SnifferometerTeam:TU-Delft/Snifferometer2012-10-27T00:39:09Z<p>MarkWeijers: </p>
<hr />
<div>{{:Team:TUDelft/Scripts}}{{:Team:TUDelft/CSSLaksh}}{{:Team:TUDelft/menu}}<br />
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<br />
</div><br />
</html><br />
<div id="contentbox" style="text-align:justify;"><br />
<br />
In order to assess the feasibility using our Snifferomyces cells as a diagnosis method, a device called "Snifferometer" was built to measure the status of fluorescence. The purpose of building such a device is to overcome the complexity, incalculability, and the high cost of using fluorescence microscope.<br />
= Construction of Snifferometer =<br />
<br />
== Scheme of the Prototype ==<br />
Figure 1 shows the concept of the mechanism of the device. <br />
<br />
The liquid is the mixture of sputum and our snifferomyces. If there are some Tuberculosis smelling cells in sputum, the snifferomyces will respond and produce fluorophores.<br />
<br />
The blue LEDs are to excite the output fluorophores in the snifferomyces yeast. Then the photodiode collects the emitted fluorescence from the yeast and convert it into voltage signal through the amplifying circuit. Thus, by reading the number of voltmeter, the status of TB smelling cells in sputum can be detected.<br />
<br />
With respect to the indicator, because at the moment sufficient data from snifferomyces is absent, we have little of what the voltage value will be with respect to certain amount of sputum and certain amount of snifferomyces. A voltmeter is used as an indicator of the prototype and we excite the cells with a blue LED, of which the threshold voltage is chosen to be between those in the case of the existence and inexistence of TB cells. When there is Tuberculosis, then light on; otherwise, light off.<br />
<br />
[[File:Scheme of snifferometer.png|270px|left|thumb|'''Figure 1''': Scheme of Snifferometer]]<br />
[[File:snifferometer device.png|250px|right|thumb|'''Figure 2''': Real prototype of Snifferometer]]<br />
<br />
</div><br />
<br />
<div id="contentbox" style="text-align:justify;"><br />
<br />
== Circuit of Snifferometer ==<br />
The photodiode converts the light intensity into small current, and op amp is needed to amplify the small current signal.<br />
<br />
[https://2012.igem.org/File:Component_sheet.PNG Click] to see the component sheet, in which the price is also included. In the sheet, the cost is around 30 euros in the case of volume production. We can make the price as little as 4 to 10 euros if considering the possible lowest price.<br />
[[File:Circuit of Snifferometer.png|580px|Right|thumb|'''Figure 3''': Circuit of Snifferometer]]<br />
</div><br />
<br />
<div id="contentbox" style="text-align:justify;"><br />
<br />
== Wavelength ==<br />
In Snifferomyces, we use EGFP as the output. By looking up in the [http://flowcyt.salk.edu/fluo.html Table of Fluorochrome], the peak excitement and emission wavelengthes of EGFP are known, which are 488nm and 507nm respectively. <br />
<br />
LEDs were chosen to emit light that would excite the EGFP efficiently, thus [http://media.digikey.com/pdf/Data%20Sheets/Optek%20PDFs/OVLFB3C7.pdf Blue LEDs] are chosen with the dominant wavelength of 470nm.<br />
<br />
The photodiode is working as a detector. Because we only care about the existence of emission light with less disturbance of excitement light. A photodiode was chosen that would detect the output with less overlap of the LED spectrum. And the responsivity spectrum of the chosen photodiode can be seen [http://www.marktechopto.com/products/datasheet/MTD5052W here]. <br />
<br />
However, it can be seen that there is still wavelength overlapping from 450 to 500nm between LED spectrum and photodiode spectrum, and photodiode basically works as detector with low accuracy in wavelength filtering. Thus a external high-pass filter is needed which has a clear cutoff frequency around 500nm. In the test, two filters were used to give a comparison:<br />
<br />
<br />
<ul><br />
<li> Filter 1: [http://www.leefilters.com/lighting/colour-details.html#768 LEE Filters 768 Egg yellow].<br />
<li> Filter 2: [http://www.leefilters.com/lighting/colour-details.html#101 LEE Filters 101 yellow].<br />
</ul><br />
<br />
</div><br />
<br />
<div id="contentbox" style="text-align:justify;"><br />
<br />
= Test =<br />
Since the fluorescence signal emitted from yeast is not very strong to reach the sensitivity of our prototype, the ''E.coli'' with TagGFP2 overexpression is then used to test the feasibility of our device. TagGFP2 has 483nm and 506nm for its excitement and emission peak wavelength which is similar with EGFP.<br />
<br />
Eppendorf tubes are used to contain the liquid. The Control sets are M9 medium with ''E.coli'', while the Test sets are M9 medium with ''E.coli'' overexpressing TagGFP2. M9 medium is used because it has low autofluorescence.<br />
<br />
[[File:Msnifferometer.jpg|420px|center|'''Figure4''': Measuring data]]<br />
<br />
<br />
[[File:Data Sniff.png|580px|middle|thumb|'''Table 1''': Measured data]]<br />
<br />
<br />
'''The data of Test sets are higher than these of Control sets, which means Snifferometer is capable to detect the glowing GFP'''. Moreover, the data also shows Filter 2 is better than Filter 1, since its passing high wavelength is overlapping more with the responsivity spectra of photodiode. <br />
<br />
'''In conclusion, the idea of building a Snifferometer is feasible'''. However, there is still room for improvement because the sensitivity of the device is not so satisfactory. Some suggested ways include:<br />
<ul><br />
<li>Reduce light intensity of background. For example, the material of Snifferometer cap can be made with black coarse surface instead of smooth blue, so the reflection of background can be reduced.<br />
<li>Increase green light intensity<br />
<li>Enhance the sensitivity of the circuit, such as increase the feedback resistor of op amp. <br />
<li>Enlarge the area of fluorescent: in the test, limited by suitable container, Eppendorf tubes were used, which has a small area to reflect and glow light. We can enhance the received signal for example by using petri dish or plate. <br />
</ul><br />
<html><br />
<br />
</div><br />
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<a href='https://2012.igem.org/Main_Page' target="_blank"><div id='logo_igem2'></div><a/></body></html></div>MarkWeijershttp://2012.igem.org/Team:TU-Delft/SnifferometerTeam:TU-Delft/Snifferometer2012-10-27T00:37:33Z<p>MarkWeijers: </p>
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In order to assess the feasibility using our Snifferomyces cells as a diagnosis method, a device called "Snifferometer" was built to measure the status of fluorescence. The purpose of building such a device is to overcome the complexity, incalculability, and the high cost of using fluorescence microscope.<br />
= Construction of Snifferometer =<br />
<br />
== Scheme of the Prototype ==<br />
Figure 1 shows the concept of the mechanism of the device. <br />
<br />
The liquid is the mixture of sputum and our snifferomyces. If there are some Tuberculosis smelling cells in sputum, the snifferomyces will respond and produce fluorophores.<br />
<br />
The blue LEDs are to excite the output fluorophores in the snifferomyces yeast. Then the photodiode collects the emitted fluorescence from the yeast and convert it into voltage signal through the amplifying circuit. Thus, by reading the number of voltmeter, the status of TB smelling cells in sputum can be detected.<br />
<br />
With respect to the indicator, because at the moment sufficient data from snifferomyces is absent, we have little of what the voltage value will be with respect to certain amount of sputum and certain amount of snifferomyces. A voltmeter is then used as an indicator of the prototype. However, the ideal indicator of a product would be a LED, of which the threshold voltage is chosen to be between those in the case of the existence and inexistence of TB cells. When there is Tuberculosis, then light on; otherwise, light off.<br />
<br />
[[File:Scheme of snifferometer.png|270px|left|thumb|'''Figure 1''': Scheme of Snifferometer]]<br />
[[File:snifferometer device.png|250px|right|thumb|'''Figure 2''': Real prototype of Snifferometer]]<br />
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== Circuit of Snifferometer ==<br />
The photodiode converts the light intensity into small current, and op amp is needed to amplify the small current signal.<br />
<br />
[https://2012.igem.org/File:Component_sheet.PNG Click] to see the component sheet, in which the price is also included. In the sheet, the cost is around 30 euros in the case of volume production. We can make the price as little as 4 to 10 euros if considering the possible lowest price.<br />
[[File:Circuit of Snifferometer.png|580px|Right|thumb|'''Figure 3''': Circuit of Snifferometer]]<br />
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== Wavelength ==<br />
In Snifferomyces, we use EGFP as the output. By looking up in the [http://flowcyt.salk.edu/fluo.html Table of Fluorochrome], the peak excitement and emission wavelengthes of EGFP are known, which are 488nm and 507nm respectively. <br />
<br />
LEDs were chosen to emit light that would excite the EGFP efficiently, thus [http://media.digikey.com/pdf/Data%20Sheets/Optek%20PDFs/OVLFB3C7.pdf Blue LEDs] are chosen with the dominant wavelength of 470nm.<br />
<br />
The photodiode is working as a detector. Because we only care about the existence of emission light with less disturbance of excitement light. A photodiode was chosen that would detect the output with less overlap of the LED spectrum. And the responsivity spectrum of the chosen photodiode can be seen [http://www.marktechopto.com/products/datasheet/MTD5052W here]. <br />
<br />
However, it can be seen that there is still wavelength overlapping from 450 to 500nm between LED spectrum and photodiode spectrum, and photodiode basically works as detector with low accuracy in wavelength filtering. Thus a external high-pass filter is needed which has a clear cutoff frequency around 500nm. In the test, two filters were used to give a comparison:<br />
<br />
<br />
<ul><br />
<li> Filter 1: [http://www.leefilters.com/lighting/colour-details.html#768 LEE Filters 768 Egg yellow].<br />
<li> Filter 2: [http://www.leefilters.com/lighting/colour-details.html#101 LEE Filters 101 yellow].<br />
</ul><br />
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= Test =<br />
Since the fluorescence signal emitted from yeast is not very strong to reach the sensitivity of our prototype, the ''E.coli'' with TagGFP2 overexpression is then used to test the feasibility of our device. TagGFP2 has 483nm and 506nm for its excitement and emission peak wavelength which is similar with EGFP.<br />
<br />
Eppendorf tubes are used to contain the liquid. The Control sets are M9 medium with ''E.coli'', while the Test sets are M9 medium with ''E.coli'' overexpressing TagGFP2. M9 medium is used because it has low autofluorescence.<br />
<br />
[[File:Msnifferometer.jpg|420px|center|'''Figure4''': Measuring data]]<br />
<br />
<br />
[[File:Data Sniff.png|580px|middle|thumb|'''Table 1''': Measured data]]<br />
<br />
<br />
'''The data of Test sets are higher than these of Control sets, which means Snifferometer is capable to detect the glowing GFP'''. Moreover, the data also shows Filter 2 is better than Filter 1, since its passing high wavelength is overlapping more with the responsivity spectra of photodiode. <br />
<br />
'''In conclusion, the idea of building a Snifferometer is feasible'''. However, there is still room for improvement because the sensitivity of the device is not so satisfactory. Some suggested ways include:<br />
<ul><br />
<li>Reduce light intensity of background. For example, the material of Snifferometer cap can be made with black coarse surface instead of smooth blue, so the reflection of background can be reduced.<br />
<li>Increase green light intensity<br />
<li>Enhance the sensitivity of the circuit, such as increase the feedback resistor of op amp. <br />
<li>Enlarge the area of fluorescent: in the test, limited by suitable container, Eppendorf tubes were used, which has a small area to reflect and glow light. We can enhance the received signal for example by using petri dish or plate. <br />
</ul><br />
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<h3home>Snifferomyces</h3home><br/><br />
<br />
<font size="2" face="sans-serif">Snifferomyces is a modular system, used in the detection of volatile compounds. It has in the membrane a <b>G-protein–coupled receptor</b> that can bind to a <b>specific signal</b>, once bound it then <b>switches on a signaling machinery</b> which <b>transmits</b> this <b>information</b> over the plasma membrane and through the cell to <b>produce a Quantitative response</b> in the form of <a href="https://2012.igem.org/Team:TU-Delft/part2"><b>fluorescence</b></a>. Using the Snifferomyces, our <b>aim</b> is to develop a <b>universal olfactory system</b> which <b>allows scientists</b> to <a href="https://2012.igem.org/Team:TU-Delft/part1#A3"><b>introduce olfactory</a> receptors in yeast with minimal effort.</b><br />
<br/><br />
<a href="2012.igem.org/Team:TU-Delft/part1#P10"><b>G-protein–coupled receptors (GPCRs)</b></a> are one of the most important classes of proteins in living organisms that allows <b>transmission of a wide variety of signals</b> over the cell membrane, between cells and over long distances in the human body, thus acting as both the <b>gatekeepers</b> and <b>molecular messengers</b> of the cell. The importance of these receptors is emphasized by the <b>Nobel prize</b> awarded in 2004 for the discoveries of <b>"odorant receptors and the organization of the olfactory system"</b> and in 2012 for <b>"studies of G-protein–coupled receptors".</b></p><br />
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<ul><li><a href="https://2012.igem.org/Team:TU-Delft/part1"> Localization of a Niacin receptor into the membrane </a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/part1#P7"> Activation of the Niacin receptor by the ligand niacin </a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/part2#A2"> Activation of the reporter by the native ligand alpha pheromone</a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/part1#P8"> Providing a platform to swap receptors, promoters and terminators more easy</a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/Modeling/StructuralModeling#youtube">Prediction of a ligand-binding niche with the niacin receptor</a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/Snifferometer">Prototype version of a yeast olfactory detector device: The Sniffer-o-meter</a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/Modeling/SingleCellModel">Deterministic and stochastic simulations of pathway model</a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/Modeling/SingleCellModel">Data fitting for deterministic pathway model</a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/HumanOutreach">To get public awareness we presented our project to a large audience by participating in several events like Llowlab (~20.000 people!) and the Floriade.</a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/HP">Case study on implementing the snifferometer as diagnostic tool for tuberculosis in developing countries</a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/Collaboration">We collaborated with teams to educate, innovate and share knowledge!</a></li><br />
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<h3home>Snifferomyces</h3home><br/><br />
<br />
<font size="2" face="sans-serif">Snifferomyces is a modular system, used in the detection of volatile compounds. It has in the membrane a <b>G-protein–coupled receptor</b> that can bind to a <b>specific signal</b>, once bound it then <b>switches on a signaling machinery</b> which <b>transmits</b> this <b>information</b> over the plasma membrane and through the cell to <b>produce a Quantitative response</b> in the form of <a href="https://2012.igem.org/Team:TU-Delft/part2"><b>fluorescence</b></a>. Using the Snifferomyces, our <b>aim</b> is to develop a <b>universal olfactory system</b> which <b>allows scientists</b> to <a href="https://2012.igem.org/Team:TU-Delft/part1#A3"><b>introduce olfactory</a> receptors in yeast with minimal effort.</b><br />
<br/><br />
<b>G-protein–coupled receptors (GPCRs)</b> are one of the most important classes of proteins in living organisms that allows <b>transmission of a wide variety of signals</b> over the cell membrane, between cells and over long distances in the human body, thus acting as both the <b>gatekeepers</b> and <b>molecular messengers</b> of the cell. The importance of these receptors is emphasized by the <b>Nobel prize</b> awarded in 2004 for the discoveries of <b>"odorant receptors and the organization of the olfactory system"</b> and in 2012 for <b>"studies of G-protein–coupled receptors".</b></p><br />
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<ul><li><a href="https://2012.igem.org/Team:TU-Delft/part1"> Localization of a Niacin receptor into the membrane </a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/part1#P7"> Activation of the Niacin receptor by the ligand niacin </a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/part2#A2"> Activation of the reporter by the native ligand alpha pheromone</a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/part1#P8"> Providing a platform to swap receptors, promoters and terminators more easy</a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/Modeling/StructuralModeling#youtube">Prediction of a ligand-binding niche with the niacin receptor</a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/Snifferometer">Prototype version of a yeast olfactory detector device: The Sniffer-o-meter</a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/Modeling/SingleCellModel">Deterministic and stochastic simulations of pathway model</a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/Modeling/SingleCellModel">Data fitting for deterministic pathway model</a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/HumanOutreach">To get public awareness we presented our project to a large audience by participating in several events like Llowlab (~20.000 people!) and the Floriade.</a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/HP">Case study on implementing the snifferometer as diagnostic tool for tuberculosis in developing countries</a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/Collaboration">We collaborated with teams to educate, innovate and share knowledge!</a></li><br />
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<div id="contentbox"> {{:Team:TU-Delft/Footer}} </div> <!-- Sponsors table. make changes in https://2012.igem.org/Team:TU-Delft/Footer --></div>MarkWeijershttp://2012.igem.org/Team:TU-DelftTeam:TU-Delft2012-10-27T00:29:57Z<p>MarkWeijers: </p>
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<h3home>Snifferomyces</h3home><br/><br />
<br />
<font size="2" face="sans-serif">Snifferomyces is a modular system, used in the detection of volatile compounds. It has in the membrane a <b>G-protein–coupled receptor</b> that can bind to a <b>specific signal</b>, once bound it then <b>switches on a signaling machinery</b> which <b>transmits</b> this <b>information</b> over the plasma membrane and through the cell to <b>produce a Quantitative response</b> in the form of <a href="https://2012.igem.org/Team:TU-Delft/part2"><b>fluorescence</b></a>. Using the Snifferomyces, our <b>aim</b> is to develop a <b>universal olfactory system</b> which <b>allows scientists</b> to <a href="https://2012.igem.org/Team:TU-Delft/part1#A3"><b>introduce olfactory</a> receptors in yeast with minimal effort.</b><br />
<br/><br />
<b>G-protein–coupled receptors (GPCRs)</b> are one of the most important classes of proteins in living organisms that allows <b>transmission of a wide variety of signals</b> over the cell membrane, between cells and over long distances in the human body, thus acting as both the <b>gatekeepers</b> and <b>molecular messengers</b> of the cell. The importance of these receptors is emphasized by the <b>Nobel prize</b> awarded in 2004 for the discoveries of <b>"odorant receptors and the organization of the olfactory system"</b> and in 2012 for <b>"studies of G-protein–coupled receptors".</b></p><br />
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<ul><li><a href="https://2012.igem.org/Team:TU-Delft/part1"> Localization of a Niacin receptor into the membrane </a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/part1#P7"> Activation of the Niacin receptor by the ligand niacin </a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/part2#A2"> Activation of the reporter by the native ligand alpha pheromone</a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/part1#P8"> Providing a platform to swap receptors, promoters and terminators more easy</a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/Snifferometer">Prototype version of a yeast olfactory detector device: The Sniffer-o-meter</a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/Modeling/SingleCellModel">Deterministic and stochastic simulations of pathway model</a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/Modeling/SingleCellModel">Data fitting for deterministic pathway model</a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/Modeling/StructuralModeling#youtube">Prediction of a ligand-binding niche with the human niacin receptor</a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/HumanOutreach">To get public awareness we presented our project to a large audience by participating in several events like Llowlab (~20.000 people!) and the Floriade.</a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/HP">Case study on implementing the snifferometer as diagnostic tool for tuberculosis in developing countries</a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/Collaboration">We collaborated with teams to educate, innovate and share knowledge!</a></li><br />
</ul><br />
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<div id="contentbox"> {{:Team:TU-Delft/Footer}} </div> <!-- Sponsors table. make changes in https://2012.igem.org/Team:TU-Delft/Footer --></div>MarkWeijershttp://2012.igem.org/Team:TU-Delft/Modeling/StructuralModelingTeam:TU-Delft/Modeling/StructuralModeling2012-10-27T00:25:47Z<p>MarkWeijers: </p>
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<h2>Summary</h2><br />
<br/><br />
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<p>In order to engineer a yeast strain that is able to detect a tuberculosis (TB) molecule like methyl nicotinate, its receptor should be designed in such a way that the molecule can act as an agonist. By modeling the olfactory receptor in silico, its biophysical and biochemical properties are investigated at the molecular level. The aim is to get a clear understanding of how a ligand binds in the receptor and how to mutate the binding niche to let it bind to methyl nicotinate more specifically.</p><br />
<br />
<p>A validated model of the human niacin receptor 1 was built and a prediction criteria was derived from experimental data of mutated variants of this receptor. Also, the human OR2AG1 receptor was successfully reproduced with its ligand docked inside. An additional hydrogen-bonding of two amino acids within the receptor was found to have an considerable influence on the conformational change properties.</p><br />
<br />
<p>It is expected that this prediction method could give more reliable models of odorant receptors and thus engineer many receptors for ligands like fruit odors, compounds exhaled by people with a disease, sense explosives and drugs.</p><br />
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<h2>Introduction</h2><br />
<br/><br />
<br />
<p>Within the Snifferomyces project, the team of iGEM TU Delft 2012 aims to develop an olfactory device to sense volatile compounds. As an application, a few snifferomyces receptors were engineered in yeast to detect molecules in the breath of a tuberculosis (TB) patient (Figure 1a-d) and to detect banana smell. </p><br />
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<a href="https://static.igem.org/mediawiki/2012/d/df/All_compounds2.png" rel="lightbox" title="AllCompounds"><br />
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<h6><b>Figure 1.</b> Chemical compounds present in the breath of a TB-patient, represented both in 2D as in 3D.[1] Methyl nicotinate (a), methyl phenylacetate (b), methyl p-anisate (c) and o-phenylanisole (d)</h6><br />
<br/><br />
<br />
<p>These included the niacin receptor 1 of Rattus Norvegicus (a.k.a. rGPR109A) and the banana receptor of Mus Musculus (a.k.a. mOlfr154). By replacing the first alpha-helix, N- and C-terminals by the ones of the I7 receptor of Rattus Norvegicus, the receptor is known to be integrated in the cell membrane [3]. These two Snifferomyces receptors, niacin receptor (NR1) and banana receptor (BR4), together with their I7 flanks, were to be modeled in molecular dynamics software, named YASARA. This way, a prediction method of the receptor activity could be implemented for use on both these receptors. However, the NR1-receptor is not specifically evolved to detect one of the TB-molecules, methyl nicotinate (Figure 1a), but niacin. In theory, the BR4-receptor can be reengineered in such a way that it could let the second TB-molecule, methyl phenylacetate (Figure 1b) act as an agonist. </p><br />
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<h3>Prediction method</h3><br />
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<br />
<br />
<p>The research group of Gerwert et al. [2] developed a method based on Molecular Dynamics (MD) simulations and experimental data, to predicted whether a molecule would act as an agonist for the hOR2AG1 receptor. Within their research, point-mutations were inserted in the gene of the hOR2AG1 receptor, in order to lower the activity hardly or dramatically. By incorporating these point-mutations in their model, they could predict whether such a mutation would indeed influence the activity of the receptor. They concluded that if a molecule would be hydrogen-bonded for >50% of 10 nanoseconds simulation time to amino acid Thr 279, and > 0% of the same time period to Ser 263 or Ser 264 in the binding niche, the receptor would be activated. This requirement was specifically targeted for molecules with an ester-configuration, just like amyl butyrate and isoamyl acetate are. </p><br />
<br />
<p>To examine whether a similar requirement could be acquired from the NR1-model, a similar analysis approach was developed by using the experimental data from the hGPR109A mutants [4]. The EC50 responses of these mutants were normalized to the response of the wildtype (WT) hOR2AG1 receptor. Some mutants were simulated together with niacin docked in their binding niche, by using Molecular Dynamics in YASARA. Instead of 10 ns simulation time, 1 ns was chosen as simulation time, mainly due to the fact that a simulation of 10 ns would take 7-8 days to complete. The results of these simulations are analyzed on hydrogen-bonding between amino acids and the ligand. That information was generated for all mutants and the WT, and thus compared to one another, to see whether there is a correlation in the hydrogen-bonded protein-ligand interaction. </p><br />
<br />
<p>In addition, all other internal hydrogen-bonds between the amino acids of the receptor were analyzed to examine whether a point-mutation has influence on these hydrogen-bonds as well, even if the point-mutation did not disrupt the binding niche directly. It was proposed that a disruption in the hydrogen-bonding between several amino acids might influence the conformational change of the receptor, thus may not activate the receptor and in turn not the G-alpha protein. Analyses of the MD results on the hGPR109A and hOR2AG1 should give an insight whether other hydrogen-bonds then the ones bound to the ligand influence the conformational change. </p><br />
<br />
<br />
<br/><br />
<br/><br />
<h3>Reengineering of binding niche</h3><br />
<br/><br />
<br />
<br />
<p>One of the main objectives the Snifferomyces project consisted of, was to engineer a receptor that has an affinity to methyl nicotinate. Because this compound and niacin are very close related, the choose to use this receptor as an template was obvious. In order to let this receptor sense methyl nicotinate with a high affinity, the binding-niche should be reprogrammed in order to let it bind. With the information on the internal hydrogen-bonding, several residues surrounding the ligand can be mutated without causing an disruption in the conformational change. </p><br />
<br />
<p>The same may hold for the BR4 receptor, which is a receptor to the ester amyl butyrate (apricot smell). The second TB-molecule, methyl phenylacetate, is also an ester. MD simulations and experimental data on the hOR2AG1 receptor [2] showed that the affinity on the ligand isoamylbenzoate (papaya smell) was increased after one point-mutation. MD simulations on the BR4-receptor with the second TB-molecule, methyl phenylacetate, should indicate whether a point-mutation for higher affinity should be necessary. </p><br />
<br />
<br/><br />
<br/><br />
<h2>Results</h2><br />
<br/><br />
<br />
<h3>BR4 Model</h3><br />
<br/><br />
<br />
<p>The BR4-model was based upon the human OR2AG1 (a.k.a. hOR2AG1) receptor, which has amyl butyrate [FIG] as an agonist. Previous research had modeled the hOR2AG1 receptor <i>in silico</i> and by using a homology modeling macro of YASARA, this model was reproduced conform the alignment done by Gerwert et.al.[2]</p><br />
<br />
<br/><br />
<a href="https://static.igem.org/mediawiki/2012/d/d4/AM.png" rel="lightbox" title="AM"><br />
<img src="https://static.igem.org/mediawiki/2012/d/d4/AM.png" name="kugroup" width="570" border="0" id="kugroup" /></a><br />
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<h6><b>Figure 2.</b> Reproduction of the hOR2AG1 binding-niche [2] by homology modeling.</h6><br />
<br/><br />
<br />
</p>After aligning the hOR2AG1 receptor with the BR4 receptor, and using the same swapping method as described for the NR1 model, the BR4-model was constructed. However, time did not permit us to execute simulations on the hOR2AG1 to test if the model would hold the same binding-niche characteristics. As soon as this model is conformed to the characteristics of the model from the paper, the BR4 sequence can be aligned with it to build the model.</p><br />
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<br/><br />
<br />
<h3>NR1 Model</h3><br />
<br/><br />
<br />
<p>The model of the NR1 receptor was based upon the predicted model of Zhang I-Tasser website [5]. In turn, this model of the human niacin receptor 1 (a.k.a. hGPR109A) was predicted by the Zhang research group. By aligning the construct of our NR1 amino acid sequence with hGPR109A (Figure 3), a homology model was built by swapping the different amino acids of hGPR109A by the ones of NR1. </p><br />
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<a href="https://static.igem.org/mediawiki/2012/8/8d/Alignment1.png" rel="lightbox" title="AM"><br />
<img src="https://static.igem.org/mediawiki/2012/8/8d/Alignment1.png" name="kugroup" width="570" border="0" id="kugroup" /></a><br />
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<h6><b>Figure 3.</b> Alignment of hGPR109A and Snifferomyces receptor NR1. </h6><br />
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<p>Several hGPR109A mutants from the data of Offermanns et al. [4] were chosen to be used in the MD simulations, which can be seen in table 1. </p><br />
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<h6><b>Table 1. </b><i>In silico</i> hydrogen-bond contacts of niacin with Homo sapiens GPR109A variants. The percentages represent frequency of hydrogen-bond contact occurrence.</h6><br />
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<a href="https://static.igem.org/mediawiki/2012/b/be/Table2_energy.png" rel="lightbox" title="Table 1"><br />
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<h6>[a] Calculated binding energy by YASARA. <br />
[b] Two criteria for receptor activation; temporary bonding to Ser 247 (> 25%) and fluctuating bonding to Arg 111 (<0%), and 1) Robust bonding to Ser 178 and Arg 251 (>50%) and 2) fluctuating and temporary bonding to Ser 178 (0-24%) and Arg 251 (25-49%) respectively with temporary bonding between Asn 45 and Ser 287. <br />
[c] <i>In vivo</i> activity qualified as EC50 response <20, otherwise inactive.<br />
[d] Mutated residue variant compared to its relevant residue. <br />
[e] NR1 receptor with ligand methyl nicotinate. </h6><br />
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<p>Table 1 shows the percentage of the total simulation time at which the amino acids are bound to the ligand. Each MD simulation was executed after an energy minimization and the specific point-mutation during a simulation time of 1 nanosecond. The active/inactive states are partly based on the classification of the responses of the hOR2AG1 mutants; every EC50 value of 20 or higher is classified as being inactive and having a low affinity. Every response below 20 is considered active.</p><br />
<br />
<p>In addition, in the paper hydrogen-bonds in time are also classified in groups; whenever there is a hydrogen-bond present for 1-24% of the simulation time, it is considered temporary bonds, fluctuating during 25-49% of the time, and robust bonds are considered when they are bound 50-100% of the simulation time. </p><br />
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<p>Analyses of table 1 shows primarily that for a hGPR109A mutant to be active, at least the protein-ligand interaction for amino acid Ser 178 and Arg 251 have to be >50, together with Ser 247 and Arg 111 , which should have a value of >25 and >0, respectively. However, this is not the case for mutant L83V, which has a very low value for its hydrogen-bonding to Ser 178 and Arg 251. When checking for the internal hydrogen-bonds, one in particular stood in correlation with this mutant and the others. The hydrogen-bonding of Asn 45 to Ser 287 can be considered robust in comparison with the other active mutants, which are <50. </p><br />
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<p>The inactive mutants all have a value of <50 for Arg 251 which classifies them as being inactive, except for N86Y and R111A. The latter mutant has no Arg 111 at all, and can be considered as being inactive by definition. The hydrogen-bonding of Arg 251 to the ligand is robust, however the protein-ligand interaction of Ser 178 very low. In comparison to the data of the mutant L83V, for the mutant N86Y to be inactive, the interaction between Ser 287 and Asn 45 should be <50, which is the case. </p><br />
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<p> Molecular Dynamics analyses of the NR1-receptor shows a result similar to mutant N86Y. However, in this case the Ser 187 - Asn 45 bonding (which is also present in the NR1 receptor) is active for 38% of the time. The boundary condition for this interaction to have influence on a weak protein-ligand bonding of Ser 178 or Arg 251, could be fixed at >25%. The interaction of methyl nicotinate to the NR1-receptor clearly shows no affinity at all, which can also be concluded from the dramatic drop in binding energy.</p><br />
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<h3>Reengineering of the NR1 receptor</h3><br />
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<br />
<p>Furthermore, another interaction of Arg 251 with its neighboring amino acid, Glu 196, was investigated. This hydrogen-bond can be considered as being very robust, whereas the bonding is always 99% or higher, except of course for mutant R251A. It seems that this mutant may have a role to play in the conformational change of the receptor, but in the absence of the amino acid the receptor is still able to be activated, but many times less than the non-mutated wildtype receptor. From this can be suggested that Arg 251 indeed may be mutated in the reprogramming assay. </p><br />
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<h2>Discussion</h2><br />
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<p>While the method of this research was mainly based upon the experimental and MD data from Gerwert et.al.[2], not all MD requirements as done by them were taken into account. The main differences were the use of MD simulation program (instead of GROMACS, YASARA was used) and the simulation time was a 10-fold lower. The higher the simulation time, the more the simulation time is similar to the real protein-ligand interaction time. </p><br />
<br />
<p>One should also take in account that the models of the hGPR109A and hOR2AG1 receptors are an approximation of reality. By doing point-mutations on these receptors, the models can be refined. For this research only a limited amount of hGPR109A mutants were available. For example, a point-mutation in Ser 247 would give more insight on the affinity of the binding niche. In addition, the NR1 and BR4 models are derivatives of the hGPR109A and hOR2AG1, respectively. The alignments included no gaps, which means that the swapping method could give a reliable representation of the Snifferomyces receptors. </p><br />
<br />
<p>To have a more accurate model of the NR1 and BR4 receptors, point-mutations on these receptors and measurement of the response to certain ligands should be implemented in further research. By running MD simulations, interesting amino acids can be selected for mutation to examine whether the expected response is generated by the receptor. </p><br />
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<p>Point-mutations on possible important amino acids like Asn 45 and Ser 287 should give an understanding whether the interaction between these two residues really act as a “fail-safe” mechanism that could still activate the G-alpha protein. Further research could give more insight into other possible residues that could be responsible for such a mechanism.</p><br />
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<p>The simulation times were a crucial part of the structural modeling in YASARA. YASARA has a friendly user's interface and has many integrated features. However, the MD simulations are optimized for use on a 8-core computer. This means that a simulation of 10 ns takes about 7-8 days to completely simulate on a 16-core computer, let alone an 8-core computer. Also, by taking in account the error probability for every simulation, this process exhausts a lot of time.</p><br />
<br />
<p>Thankfully, the <a href="https://www.sara.nl/">SARA institute</a> was willing to help us and assist us in setting up the environment to use their HPC Cloud Server remotely from our office at Delft University. This gave us the opportunity to execute our simulations on a configurable amount of cores. By testing what amount of cores was the fastest to use (but not necessarily the most efficient) short simulations on 4, 8, 12, 16, 18, 20 and 24 cores were performed. The outcome was in favor of the 16-core computer. This means that a simulation of 10 ns would take 7-8 days. In contrast, a simulation of 1 nanosecond took about 2-2.5 days.</p>. <br />
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<h2>Follow-up</h2><br />
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<p>Some additional ideas on structural modeling had to be given a lower priority. An interesting approach would be to engineer an olfactory receptor for one of the other three chemical compounds (Figure 1b-d) which are found in the breath of a tuberculosis patient. In the same manner, the reprogramming of the binding niche could allow different derivatives of a certain compound (methyl isonicotinate instead of methyl nicotinate) allow to bind, of which a kind of universality could be investigated to predict a configuration of the binding niche to any ligand.</p><br />
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<p>Another follow-up would be to investigate <i>in silico</i> how large the conformational change of the receptor is, and see how this correlates to the hydrogen bonding, RSMD and energy. This would require data on simulations done with and without a ligand. This could eventually lead to a value for the dissociation rate between the G-alpha protein and the receptor.</p><br />
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<p>Because the hOR2AG1 is a receptor to a fruity molecule, there is a potential that it could sense other odors van fruits that hold an ester-configuration as well. Many of these odors are derivatives of each other, so the BR4 receptor could be reengineered in such a way that it can sense any fruity molecule.</p><br />
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<h2>References</h2><br />
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<br />
<h6><br />
[1] Syhre M, Chambers ST (2008) The scent of Mycobacterium tuberculosis. Tuberculosis. 88:317–323<br />
[2] Gelis L, Wolf S, Hatt H, Neuhaus EM, Gerwert K (2012) Prediction of a Ligand-Binding Niche within a Human Olfactory Receptor by Combining Site-Directed Mutagenesis with Dynamic Homology Modeling. Angew. Chem. Int. Ed. 51:1274-1278<br />
[3] Jasmina Minic, Marie-annick Persuy, Elodie Godel, Josiane Aioun, Ian Connerton, Roland Salesse, Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening, FEBS Journal (2005)<br />
[4] Tunaru S, Lättig J, Kero J, Krause G, Offermanns S (2005) Characterization of Determinants of Ligand Binding to the Nicotinic Acid Receptor GPR109A (HM74A/PUMA-G). Mol Pharmacol. 68:1271-1280<br />
[5]J Zhang, Y Zhang. GPCR-ITASSER: A new composite algorithm for G protein-coupled receptor structure prediction and the application on human genome. 2011</h6><br />
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<h3home>Snifferomyces</h3home><br/><br />
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<font size="2" face="sans-serif">Snifferomyces is a modular system, used in the detection of volatile compounds. It has in the membrane a <b>G-protein–coupled receptor</b> that can bind to a <b>specific signal</b>, once bound it then <b>switches on a signaling machinery</b> which <b>transmits</b> this <b>information</b> over the plasma membrane and through the cell to <b>produce a Quantitative response</b> in the form of <a href="https://2012.igem.org/Team:TU-Delft/part2"><b>fluorescence</b></a>. Using the Snifferomyces, our <b>aim</b> is to develop a <b>universal olfactory system</b> which <b>allows scientists</b> to <a href="https://2012.igem.org/Team:TU-Delft/part1#A3"><b>introduce olfactory</a> receptors in yeast with minimal effort.</b><br />
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<b>G-protein–coupled receptors (GPCRs)</b> are one of the most important classes of proteins in living organisms that allows <b>transmission of a wide variety of signals</b> over the cell membrane, between cells and over long distances in the human body, thus acting as both the <b>gatekeepers</b> and <b>molecular messengers</b> of the cell. The importance of these receptors is emphasized by the <b>Nobel prize</b> awarded in 2004 for the discoveries of <b>"odorant receptors and the organization of the olfactory system"</b> and in 2012 for <b>"studies of G-protein–coupled receptors".</b></p><br />
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<ul><li><a href="https://2012.igem.org/Team:TU-Delft/part1"> Localization of a Niacin receptor into the membrane </a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/part1#P7"> Activation of the Niacin receptor by the ligand niacin </a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/part2#A2"> Activation of the reporter by the native ligand alpha pheromone</a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/part1#P8"> Providing a platform to swap receptors, promoters and terminators more easy</a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/Snifferometer">Prototype version of a yeast olfactory detector device: The Sniffer-o-meter</a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/Modeling/SingleCellModel">Deterministic and stochastic simulations and property analyses of pathway model</a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/Modeling/SingleCellModel">Data fitting for deterministic pathway model</a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/Modeling/StructuralModeling#A1">Prediction of a ligand-binding niche within the human niacin receptor 1 with Molecular Dynamics simulations </a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/HumanOutreach">To find stake holders and create awareness among our project, we presented our project to a very large audience by participating in several events like Llowlab on Lowlands and the Floriade.</a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/HP">Our main goal is to innovate a diagnostic tool for tuberculosis, one of the major health issues in the world</a></li><br />
<li><a href="https://2012.igem.org/Team:TU-Delft/Collaboration">We have provided the iGEM Paris 2012 team two Biobricks. Together with the Amsterdam team we were able to organize a crash course on the area of synthetic biology and we were honored to attend the LIFE-symposium with the Cambrigde 2010-team.</a></li><br />
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<div id="contentbox"> {{:Team:TU-Delft/Footer}} </div> <!-- Sponsors table. make changes in https://2012.igem.org/Team:TU-Delft/Footer --></div>MarkWeijershttp://2012.igem.org/Team:TU-Delft/part3Team:TU-Delft/part32012-10-27T00:07:35Z<p>MarkWeijers: </p>
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<h2>Content</h2> <br />
<a href="#P10"> Introduction</a><br><br />
<a href="#P9">Parts </a><br><br />
<a href="#P8">Results </a><br><br />
<a href="#P7"> Conclusions</a><br><br />
<a href="#P9">References</a><br><br />
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<a name="P10"> <br><h2>Introduction</h2> </a><br />
<h3>Yeast with olfactory receptor+reporter=Snifferomyces</h3><br />
<p>By combining the olfactory receptor and the <i>FUS1pr-EGFP</i> reporter, a complete yeast olfactory system is obtained. If the corresponding ligand binds to the receptor the <i>FAR1</i> promoter is turned on and the EGFP is expressed. This EGFP signal can be read out by a fluorescence meter. If the olfactory system will be implemented as a diagnostics tool in developing countries, the EGFP reporter should be changed by a visible reporter.<br/><br />
<h3>Growth arrest</h3><br />
Besides the induction of the <i>FUS1</i> promoter the cells also go in growth arrest mediated by the <i>FAR1</i> promoter. However it is undesirable that the cells stop growing once they respond to a ligand. Therefore it is needed to knock out the <i>FAR1</i> promoter. Therefore we characterized the knockout of the <i>FAR1</i> promoter. <br/><br />
<h3>Increasing sensitivity</h3><br />
To optimize the signal transduction from the receptor to the downstream cascade, a mammalian alpha subunit can be introduced which has affinity with the RI7-receptor [1]. For this, making a knockout of the native <i>GPA1</i> gene is needed in order to let the subunit work as substitute. In the results we describe how we generated a knockout in yeast. In future work the alpha subunit should be expressed and characterized. </p><br />
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<a name="P9"> <h2>Parts</h2> </a> <br />
<p>The receptor constructs and the reporter constructs are combined to have one complete olfactory system. The following biobricks are created:</p><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/d/d2/GPR109A_snifferomyces.jpg" width="620" /><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775005 " target="_blank">BBa_K775005</a><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/83/OLF154_snifferomyces.jpg" width="620"/><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775006 " target="_blank">BBa_K775006</a><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/1/16/ORIG1_snifferomyces.jpg" width="620"/><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775007 " target="_blank">BBa_K775007</a><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/3/3f/Odr10_snifferomyces.jpg" width="620"/><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775008 " target="_blank">BBa_K775008</a><br/><br/><br />
<a name="P8"><h2>Results</h2></a><br />
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<h3>Characterization <i>Far1</i> Knockout </h3><br />
<p>The <i>FAR1</i> has been knocked out to prevent growth arrest once a ligand has been added. To test this we have measured the optical density of Wild Type and Knockout strains with and without alpha feromones. <br/><br />
<br/><br />
The results seem to show that there is a reduction in growth in both strains after the addition of alpha-feromones, although the <i>dfar1</i>-strain shows less reduction. </br><br />
Another remarkable feature is that the <i>Δfar1</i>-strain generally appears to grow faster than the Wild-type strain. A possible explanation is that the gene for growth-arrest has been knocked out. The <i>FAR1</i> is not only related to the mating response, but is also a control in the mitotic cycle of budding yeast. [2] </br><br />
</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/6/66/Groeicurve.jpg" align="middle" width="100%"><br />
<br />
<br />
<h3>Transformations</i></h3><br />
<p>Yeast strains were successfully transformed with the <i>GPR109A</i> receptor and output <br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775005" target="_blank">BBa_K775005</a>, called <b>NR1</b> (niacin reporter). The <i>RI7-ODR10</i> receptor and output <br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775008" target="_blank">BBa_K775008</a>. <i>Olfr154</i> receptor and output <br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775010" target="_blank">BBa_K775010</a>, called <b>BR1</b> (banana reporter 1) and <i>RI7-OR1G1</i> receptor and output <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775011">BBa_K775011</a>, called <b>BR2</b> (banana reporter 2).</p><br />
<br/><br />
<p><br />
<h3>Niacin Snifferomyces</h3><br/><br />
<h6>Setup</h6><br/><br />
In order to test the yeast NR1 cells with niacin receptor and the receptor inducible GFP reporter we induced the cells with niacin or alpha pheromone and measured the cellular response through flow cytometry, measuring the EGFP signal intensity.<br />
<h6>Outcome</h6><br/><br />
Flow cytometry data of +NR1 cells induced with niacin or alpha pheromones is shown.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2012/c/c4/Flow_Cytometry_Snifferomyces_NR1_graph.png" width="600"><br />
<p><br />
Here we see that addition of receptor ligand doesn’t induce an increased light response in comparison with alpha induced cells. Possible explanations are:<br />
<ul><li>A too small subset of the yeast receptors is localized in the membrane.</li><br />
<li>The affinity of the receptor with the alpha subunit is too low.</li><br />
<br />
<h3>Isoamyl acetate Snifferomyces</h3><br />
<h6>Setup</h6><br />
<h6>Outcome</h6><br />
<br />
<a name="P7"> <h2>Conclusions</h2> </a><br />
<p>Four olfactory system biobricks were added to the registry. The light response when adding ligand was seen with single cells, but didn't lead to statistical significant data. Further we observed that expression of receptors led to decreased fitness and non-typical morphology.</p><br/><br />
<a name="P8"> <h2>Recommendations</h2> </a><br />
To increase expression and light intensity of the cells in response to a ligand the following conditions can be tested:<br />
<ul><li> Make the receptor expression time as short as possible. Use an inducible promoter or make stocks as soon as possible after transformation </li><br />
<li> There are indications that succesful expression can be altered by decreasing the temperature during receptor expression. When we tested this the growth rate was slow and therefore the expression time became too long. An inducible promoter can solve this problem too.</li><br />
<li> The affinity towards the downstream pathway could be increased when the pheromone binding subunit alpha is knocked out (<i>dGPA1</i>) and replaced by a mammalian one (typically <a href="http://www.ncbi.nlm.nih.gov/protein/47271350?report=genbank&log$=prottop&blast_rank=1&RID=8NZM5VY001R">NP_034437.1</a> is used. We didn't have the time to test this hypothesis, further investigation is needed!</li></ul><br/><br />
<br />
<a name="P9"><h2>References</h2> </a><br />
<h6>[1] Jasmina Minic, Marie-annick Persuy, Elodie Godel, Josiane Aioun, Ian Connerton, Roland Salesse, Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening, FEBS Journal (2005)</h6><br />
<h6>[2] "A cell sizer network involving Cln3 and Far1 controls entrance into S phase in the mitotic cycle of budding yeast" Alberghina et al. November 1, 2004 // JCB vol. 167 no. 3 433-443 </h6><br />
<br />
<a name="P8"> <h2>Recommendation</h2></a><br />
<br />
<br />
</div><br />
<img src="https://static.igem.org/mediawiki/igem.org/3/37/Footer_2.jpg" align="middle" width="690"><br />
<a href='https://2012.igem.org/Main_Page' target="_blank"><div id='logo_igem2'></div><a/></body></html></div>MarkWeijershttp://2012.igem.org/Team:TU-Delft/part3Team:TU-Delft/part32012-10-27T00:06:35Z<p>MarkWeijers: </p>
<hr />
<div>{{:Team:TUDelft/Scripts}}{{:Team:TUDelft/CSS}}{{:Team:TUDelft/menu}}<br />
<html><br />
<head><title>Receptor</title></head><br />
<body><br />
<div style="height:70px; width:100%;"></div><br />
<div id="logo_ed"><a href="https://2012.igem.org/Team:TU-Delft" 'onfocus=this.blur()'><img src="https://static.igem.org/mediawiki/igem.org/8/88/Logoigemklein.png" border="0" width="100" height="100"></a></div><br />
<br />
<div id="contentbox" style="text-align:justify;"><br />
<img src="https://static.igem.org/mediawiki/igem.org/e/e4/Snifferomyces.jpg" align="middle" width="100%"><br />
<br />
<br />
<h2>Content</h2> <br />
<a href="#P10"> Introduction</a><br><br />
<a href="#P9">Parts </a><br><br />
<a href="#P8">Results </a><br><br />
<a href="#P7"> Conclusions</a><br><br />
<a href="#P9">References</a><br><br />
<br />
<a name="P10"> <br><h2>Introduction</h2> </a><br />
<h3>Yeast with olfactory receptor+reporter=Snifferomyces</h3><br />
<p>By combining the olfactory receptor and the <i>FUS1pr-EGFP</i> reporter, a complete yeast olfactory system is obtained. If the corresponding ligand binds to the receptor the <i>FAR1</i> promoter is turned on and the EGFP is expressed. This EGFP signal can be read out by a fluorescence meter. If the olfactory system will be implemented as a diagnostics tool in developing countries, the EGFP reporter should be changed by a visible reporter.<br/><br />
<h3>Growth arrest</h3><br />
Besides the induction of the <i>FUS1</i> promoter the cells also go in growth arrest mediated by the <i>FAR1</i> promoter. However it is undesirable that the cells stop growing once they respond to a ligand. Therefore it is needed to knock out the <i>FAR1</i> promoter. Therefore we characterized the knockout of the <i>FAR1</i> promoter. <br/><br />
<h3>Increasing sensitivity</h3><br />
To optimize the signal transduction from the receptor to the downstream cascade, a mammalian alpha subunit can be introduced which has affinity with the RI7-receptor [1]. For this, making a knockout of the native <i>GPA1</i> gene is needed in order to let the subunit work as substitute. In the results we describe how we generated a knockout in yeast. In future work the alpha subunit should be expressed and characterized. </p><br />
<br />
<br />
<a name="P9"> <h2>Parts</h2> </a> <br />
<p>The receptor constructs and the reporter constructs are combined to have one complete olfactory system. The following biobricks are created:</p><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/d/d2/GPR109A_snifferomyces.jpg" width="620" /><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775005 " target="_blank">BBa_K775005</a><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/83/OLF154_snifferomyces.jpg" width="620"/><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775006 " target="_blank">BBa_K775006</a><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/1/16/ORIG1_snifferomyces.jpg" width="620"/><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775007 " target="_blank">BBa_K775007</a><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/3/3f/Odr10_snifferomyces.jpg" width="620"/><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775008 " target="_blank">BBa_K775008</a><br/><br/><br />
<a name="P8"><h2>Results</h2></a><br />
<br />
<h3>Characterization <i>Far1</i> Knockout </h3><br />
<p>The <i>FAR1</i> has been knocked out to prevent growth arrest once a ligand has been added. To test this we have measured the optical density of Wild Type and Knockout strains with and without alpha feromones. <br/><br />
<br/><br />
The results seem to show that there is a reduction in growth in both strains after the addition of alpha-feromones, although the <i>Δfar1</i>-strain shows less reduction. </br><br />
Another remarkable feature is that the <i>Δfar1</i>-strain generally appears to grow faster than the Wild-type strain. A possible explanation is that the gene for growth-arrest has been knocked out. The <i>FAR1</i> is not only related to the mating response, but is also a control in the mitotic cycle of budding yeast. [2] </br><br />
</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/6/66/Groeicurve.jpg" align="middle" width="100%"><br />
<br />
<br />
<h3>Transformations</i></h3><br />
<p>Yeast strains were successfully transformed with the <i>GPR109A</i> receptor and output <br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775005" target="_blank">BBa_K775005</a>, called <b>NR1</b> (niacin reporter). The <i>RI7-ODR10</i> receptor and output <br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775008" target="_blank">BBa_K775008</a>. <i>Olfr154</i> receptor and output <br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775010" target="_blank">BBa_K775010</a>, called <b>BR1</b> (banana reporter 1) and <i>RI7-OR1G1</i> receptor and output <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775011">BBa_K775011</a>, called <b>BR2</b> (banana reporter 2).</p><br />
<br/><br />
<p><br />
<h3>Niacin Snifferomyces</h3><br/><br />
<h6>Setup</h6><br/><br />
In order to test the yeast NR1 cells with niacin receptor and the receptor inducible GFP reporter we induced the cells with niacin or alpha pheromone and measured the cellular response through flow cytometry, measuring the EGFP signal intensity.<br />
<h6>Outcome</h6><br/><br />
Flow cytometry data of +NR1 cells induced with niacin or alpha pheromones is shown.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2012/c/c4/Flow_Cytometry_Snifferomyces_NR1_graph.png" width="600"><br />
<p><br />
Here we see that addition of receptor ligand doesn’t induce an increased light response in comparison with alpha induced cells. Possible explanations are:<br />
<ul><li>A too small subset of the yeast receptors is localized in the membrane.</li><br />
<li>The affinity of the receptor with the alpha subunit is too low.</li><br />
<br />
<h3>Isoamyl acetate Snifferomyces</h3><br />
<h6>Setup</h6><br />
<h6>Outcome</h6><br />
<br />
<a name="P7"> <h2>Conclusions</h2> </a><br />
<p>Four olfactory system biobricks were added to the registry. The light response when adding ligand was seen with single cells, but didn't lead to statistical significant data. Further we observed that expression of receptors led to decreased fitness and non-typical morphology.</p><br/><br />
<a name="P8"> <h2>Recommendations</h2> </a><br />
To increase expression and light intensity of the cells in response to a ligand the following conditions can be tested:<br />
<ul><li> Make the receptor expression time as short as possible. Use an inducible promoter or make stocks directly as soon as possible after transformation </li><br />
<li> There are indications that succesful expression can be altered by decreasing the temperature during receptor expression. When we tested this the growth rate was slow and therefore the expression time became too long. An inducible promoter can solve this problem too.</li><br />
<li> The affinity towards the downstream pathway could be increased when the pheromone binding subunit alpha is knocked out (<i>dGPA1</i>) and replaced by a mammalian one (typically <a href="http://www.ncbi.nlm.nih.gov/protein/47271350?report=genbank&log$=prottop&blast_rank=1&RID=8NZM5VY001R">NP_034437.1</a> is used. We didn't have the time to test this hypothesis, further investigation is needed!</li></ul><br/><br />
<br />
<a name="P9"><h2>References</h2> </a><br />
<h6>[1] Jasmina Minic, Marie-annick Persuy, Elodie Godel, Josiane Aioun, Ian Connerton, Roland Salesse, Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening, FEBS Journal (2005)</h6><br />
<h6>[2] "A cell sizer network involving Cln3 and Far1 controls entrance into S phase in the mitotic cycle of budding yeast" Alberghina et al. November 1, 2004 // JCB vol. 167 no. 3 433-443 </h6><br />
<br />
<a name="P8"> <h2>Recommendation</h2></a><br />
<br />
<br />
</div><br />
<img src="https://static.igem.org/mediawiki/igem.org/3/37/Footer_2.jpg" align="middle" width="690"><br />
<a href='https://2012.igem.org/Main_Page' target="_blank"><div id='logo_igem2'></div><a/></body></html></div>MarkWeijershttp://2012.igem.org/Team:TU-Delft/part3Team:TU-Delft/part32012-10-26T22:36:28Z<p>MarkWeijers: </p>
<hr />
<div>{{:Team:TUDelft/Scripts}}{{:Team:TUDelft/CSS}}{{:Team:TUDelft/menu}}<br />
<html><br />
<head><title>Receptor</title></head><br />
<body><br />
<div style="height:70px; width:100%;"></div><br />
<div id="logo_ed"><a href="https://2012.igem.org/Team:TU-Delft" 'onfocus=this.blur()'><img src="https://static.igem.org/mediawiki/igem.org/8/88/Logoigemklein.png" border="0" width="100" height="100"></a></div><br />
<br />
<div id="contentbox" style="text-align:justify;"><br />
<img src="https://static.igem.org/mediawiki/igem.org/e/e4/Snifferomyces.jpg" align="middle" width="100%"><br />
<br />
<br />
<h2>Content</h2> <br />
<a href="#P10"> Introduction</a><br><br />
<a href="#P9">Parts </a><br><br />
<a href="#P8">Results </a><br><br />
<a href="#P7"> Conclusions</a><br><br />
<a href="#P9">References</a><br><br />
<br />
<a name="P10"> <br><h2>Introduction</h2> </a><br />
<h3>Yeast with olfactory receptor+reporter=Snifferomyces</h3><br />
<p>By combining the olfactory receptor and the <i>FUS1pr-EGFP</i> reporter, a complete yeast olfactory system is obtained. If the corresponding ligand binds to the receptor the <i>FAR1</i> promoter is turned on and the EGFP is expressed. This EGFP signal can be read out by a fluorescence meter. If the olfactory system will be implemented as a diagnostics tool in developing countries, the EGFP reporter should be changed by a visible reporter.<br/><br />
<h3>Growth arrest</h3><br />
Besides the induction of the <i>FUS1</i> promoter the cells also go in growth arrest mediated by the <i>FAR1</i> promoter. However it is undesirable that the cells stop growing once they respond to a ligand. Therefore it is needed to knock out the <i>FAR1</i> promoter. Therefore we characterized the knockout of the <i>FAR1</i> promoter. <br/><br />
<h3>Increasing sensitivity</h3><br />
To optimize the signal transduction from the receptor to the downstream cascade, a mammalian alpha subunit can be introduced which has affinity with the RI7-receptor [1]. For this, making a knockout of the native <i>GPA1</i> gene is needed in order to let the subunit work as substitute. In the results we describe how we generated a knockout in yeast. In future work the alpha subunit should be expressed and characterized. </p><br />
<br />
<br />
<a name="P9"> <h2>Parts</h2> </a> <br />
<p>The receptor constructs and the reporter constructs are combined to have one complete olfactory system. The following biobricks are created:</p><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/d/d2/GPR109A_snifferomyces.jpg" width="620" /><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775005 " target="_blank">BBa_K775005</a><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/83/OLF154_snifferomyces.jpg" width="620"/><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775006 " target="_blank">BBa_K775006</a><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/1/16/ORIG1_snifferomyces.jpg" width="620"/><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775007 " target="_blank">BBa_K775007</a><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/3/3f/Odr10_snifferomyces.jpg" width="620"/><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775008 " target="_blank">BBa_K775008</a><br/><br/><br />
<a name="P8"><h2>Results</h2></a><br />
<br />
<h3>Characterization <i>Far1</i> Knockout </h3><br />
<p>The <i>FAR1</i> has been knocked out to prevent growth arrest once a ligand has been added. To test this we have measured the optical density of Wild Type and Knockout strains with and without alpha feromones. <br/><br />
<br/><br />
The results seem to show that there is a reduction in growth in both strains after the addition of alpha-feromones, although the <i>Δfar1</i>-strain shows less reduction. </br><br />
Another remarkable feature is that the <i>Δfar1</i>-strain generally appears to grow faster than the Wild-type strain. A possible explanation is that the gene for growth-arrest has been knocked out. The <i>FAR1</i> is not only related to the mating response, but is also a control in the mitotic cycle of budding yeast. [2] </br><br />
</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/6/66/Groeicurve.jpg" align="middle" width="100%"><br />
<br />
<br />
<h3>Transformations</i></h3><br />
<p>Yeast strains were successfully transformed with the <i>GPR109A</i> receptor and output <br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775005" target="_blank">BBa_K775005</a>, called <b>NR1</b> (nicotinic acid reporter). The <i>RI7-ODR10</i> receptor and output <br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775008" target="_blank">BBa_K775008</a>. <i>Olfr154</i> receptor and output <br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775010" target="_blank">BBa_K775010</a>, called <b>BR1</b> (banana reporter 1) and <i>RI7-OR1G1</i> receptor and output <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775011">BBa_K775011</a>, called <b>BR2</b> (banana reporter 2).</p><br />
<br/><br />
<p><br />
<h3>Niacin Snifferomyces</h3><br/><br />
<h6>Setup</h6><br/><br />
In order to test the yeast NR1 cells with nicotinic acid receptor and the receptor inducible GFP reporter we induced the cells with nicotinic acid or alpha pheromone and measured the cellular response through flow cytometry.<br />
<h6>Outcome</h6><br/><br />
Flow cytometry data of +NR1 cells induced with nicotinic acid or alpha pheromones is shown.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2012/c/c4/Flow_Cytometry_Snifferomyces_NR1_graph.png" width="600"><br />
<p><br />
Here we see that addition of receptor ligand doesn’t induce a response in comparison with alpha induced cells. Possible explanations are:<br />
<ul><li>A too small subset of the yeast receptors is localized in the membrane.</li><br />
<li>The affinity of the receptor with the alpha subunit is too low.</li><br />
<br />
<h3>Isoamyl acetate Snifferomyces</h3><br />
<h6>Setup</h6><br />
<h6>Outcome</h6><br />
<br />
<a name="P7"> <h2>Conclusions</h2> </a><br />
<p>Four olfactory system biobricks were added to the registry. </p><br/><br />
<a name="P9"><h2>References</h2> </a><br />
<h6>[1] Jasmina Minic, Marie-annick Persuy, Elodie Godel, Josiane Aioun, Ian Connerton, Roland Salesse, Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening, FEBS Journal (2005)</h6><br />
<h6>[2] "A cell sizer network involving Cln3 and Far1 controls entrance into S phase in the mitotic cycle of budding yeast" Alberghina et al. November 1, 2004 // JCB vol. 167 no. 3 433-443 </h6><br />
<br />
</div><br />
<img src="https://static.igem.org/mediawiki/igem.org/3/37/Footer_2.jpg" align="middle" width="690"><br />
<a href='https://2012.igem.org/Main_Page' target="_blank"><div id='logo_igem2'></div><a/></body></html></div>MarkWeijershttp://2012.igem.org/Team:TU-Delft/part3Team:TU-Delft/part32012-10-26T22:33:43Z<p>MarkWeijers: </p>
<hr />
<div>{{:Team:TUDelft/Scripts}}{{:Team:TUDelft/CSS}}{{:Team:TUDelft/menu}}<br />
<html><br />
<head><title>Receptor</title></head><br />
<body><br />
<div style="height:70px; width:100%;"></div><br />
<div id="logo_ed"><a href="https://2012.igem.org/Team:TU-Delft" 'onfocus=this.blur()'><img src="https://static.igem.org/mediawiki/igem.org/8/88/Logoigemklein.png" border="0" width="100" height="100"></a></div><br />
<br />
<div id="contentbox" style="text-align:justify;"><br />
<img src="https://static.igem.org/mediawiki/igem.org/e/e4/Snifferomyces.jpg" align="middle" width="100%"><br />
<br />
<br />
<h2>Content</h2> <br />
<a href="#P10"> Introduction</a><br><br />
<a href="#P9">Parts </a><br><br />
<a href="#P8">Results </a><br><br />
<a href="#P7"> Conclusions</a><br><br />
<a href="#P9">References</a><br><br />
<br />
<a name="P10"> <br><h2>Introduction</h2> </a><br />
<h3>Yeast with olfactory receptor+reporter=Snifferomyces</h3><br />
<p>By combining the olfactory receptor and the <i>FUS1pr-EGFP</i> reporter, a complete yeast olfactory system is obtained. If the corresponding ligand binds to the receptor the <i>FAR1</i> promoter is turned on and the EGFP is expressed. This EGFP signal can be read out by a fluorescence meter. If the olfactory system will be implemented as a diagnostics tool in developing countries, the EGFP reporter should be changed by a visible reporter.<br/><br />
<h3>Growth arrest</h3><br />
Besides the induction of the <i>FUS1</i> promoter the cells also go in growth arrest mediated by the <i>FAR1</i> promoter. However it is undesirable that the cells stop growing once they respond to a ligand. Therefore it is needed to knock out the <i>FAR1</i> promoter. Therefore we characterized the knockout of the <i>FAR1</i> promoter. <br/><br />
<h3>Increasing sensitivity</h3><br />
To optimize the signal transduction from the receptor to the downstream cascade, a mammalian alpha subunit can be introduced which has affinity with the RI7-receptor [1]. For this, making a knockout of the native <i>GPA1</i> gene is needed in order to let the subunit work as substitute. In the results we describe how we generated a knockout in yeast. In future work the alpha subunit should be expressed and characterized. </p><br />
<br />
<br />
<a name="P9"> <h2>Parts</h2> </a> <br />
<p>The receptor constructs and the reporter constructs are combined to have one complete olfactory system. The following biobricks are created:</p><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/d/d2/GPR109A_snifferomyces.jpg" width="620" /><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775005 " target="_blank">BBa_K775005</a><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/83/OLF154_snifferomyces.jpg" width="620"/><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775006 " target="_blank">BBa_K775006</a><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/1/16/ORIG1_snifferomyces.jpg" width="620"/><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775007 " target="_blank">BBa_K775007</a><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/3/3f/Odr10_snifferomyces.jpg" width="620"/><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775008 " target="_blank">BBa_K775008</a><br/><br/><br />
<a name="P8"><h2>Results</h2></a><br />
<br />
<h3>Characterization <i>Far1</i> Knockout </h3><br />
<p>The <i>FAR1</i> has been knocked out to prevent growth arrest once a ligand has been added. To test this we have measured the optical density of Wild Type and Knockout strains with and without alpha feromones. <br/><br />
<br/><br />
The results seem to show that there is a reduction in growth in both strains after the addition of alpha-feromones, although the <i>Δfar1</i>-strain shows less reduction. </br><br />
Another remarkable feature is that the <i>Δfar1</i>-strain generally appears to grow faster than the Wild-type strain. A possible explanation is that the gene for growth-arrest has been knocked out. The <i>FAR1</i> is not only related to the mating response, but is also a control in the mitotic cycle of budding yeast. [2] </br><br />
</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/6/66/Groeicurve.jpg" align="middle" width="100%"><br />
<br />
<br />
<h3>Transformations</i></h3><br />
<p>Yeast strains were successfully transformed with the <i>GPR109A</i> receptor and output <br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775005" target="_blank">BBa_K775005</a>, called <b>NR1</b> (nicotinic acid reporter). The <i>RI7-ODR10</i> receptor and output <br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775008" target="_blank">BBa_K775008</a>. <i>Olfr154</i> receptor and output <br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775010" target="_blank">BBa_K775010</a>, called <b>BR1</b> (banana reporter 1) and <i>RI7-OR1G1</i> receptor and output <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775011">BBa_K775011</a>, called <b>BR2</b> (banana reporter 2).</p><br />
<br/><br />
<p><br />
<h3>Niacin Snifferomyces</h3><br/><br />
<h6>Setup</h6><br/><br />
In order to test the yeast NR1 cells with nicotinic acid receptor and the receptor inducible GFP reporter we induced the cells with nicotinic acid or alpha pheromone and measured the cellular response through flow cytometry.<br />
<h6>Outcome</h6><br/><br />
Flow cytometry data of +NR1 cells induced with nicotinic acid or alpha pheromones is shown.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2012/c/c4/Flow_Cytometry_Snifferomyces_NR1_graph.png" width="600"><br />
<p><br />
Here we see that addition of receptor ligand doesn’t induce a response in comparison with alpha induced cells. Also in terms of a cutoff comparison in terms of percentages this seems to be the case. Possible explanations are:<br />
<ul><li>A too small subset of the yeast receptors is localized in the membrane.</li><br />
<li>The affinity of the receptor with the alpha subunit is too low.</li><br />
<br />
<br />
<br />
<h3>Isoamyl acetate Snifferomyces</h3><br />
<h6>Setup</h6><br />
<h6>Outcome</h6><br />
<br />
<a name="P7"> <h2>Conclusions</h2> </a><br />
<p>Four olfactory system biobricks were added to the registry. </p><br/><br />
<a name="P9"><h2>References</h2> </a><br />
<h6>[1] Jasmina Minic, Marie-annick Persuy, Elodie Godel, Josiane Aioun, Ian Connerton, Roland Salesse, Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening, FEBS Journal (2005)</h6><br />
<h6>[2] "A cell sizer network involving Cln3 and Far1 controls entrance into S phase in the mitotic cycle of budding yeast" Alberghina et al. November 1, 2004 // JCB vol. 167 no. 3 433-443 </h6><br />
<br />
</div><br />
<img src="https://static.igem.org/mediawiki/igem.org/3/37/Footer_2.jpg" align="middle" width="690"><br />
<a href='https://2012.igem.org/Main_Page' target="_blank"><div id='logo_igem2'></div><a/></body></html></div>MarkWeijershttp://2012.igem.org/Team:TU-Delft/part3Team:TU-Delft/part32012-10-26T22:33:13Z<p>MarkWeijers: </p>
<hr />
<div>{{:Team:TUDelft/Scripts}}{{:Team:TUDelft/CSS}}{{:Team:TUDelft/menu}}<br />
<html><br />
<head><title>Receptor</title></head><br />
<body><br />
<div style="height:70px; width:100%;"></div><br />
<div id="logo_ed"><a href="https://2012.igem.org/Team:TU-Delft" 'onfocus=this.blur()'><img src="https://static.igem.org/mediawiki/igem.org/8/88/Logoigemklein.png" border="0" width="100" height="100"></a></div><br />
<br />
<div id="contentbox" style="text-align:justify;"><br />
<img src="https://static.igem.org/mediawiki/igem.org/e/e4/Snifferomyces.jpg" align="middle" width="100%"><br />
<br />
<br />
<h2>Content</h2> <br />
<a href="#P10"> Introduction</a><br><br />
<a href="#P9">Parts </a><br><br />
<a href="#P8">Results </a><br><br />
<a href="#P7"> Conclusions</a><br><br />
<a href="#P9">References</a><br><br />
<br />
<a name="P10"> <br><h2>Introduction</h2> </a><br />
<h3>Yeast with olfactory receptor+reporter=Snifferomyces</h3><br />
<p>By combining the olfactory receptor and the <i>FUS1pr-EGFP</i> reporter, a complete yeast olfactory system is obtained. If the corresponding ligand binds to the receptor the <i>FAR1</i> promoter is turned on and the EGFP is expressed. This EGFP signal can be read out by a fluorescence meter. If the olfactory system will be implemented as a diagnostics tool in developing countries, the EGFP reporter should be changed by a visible reporter.<br/><br />
<h3>Growth arrest</h3><br />
Besides the induction of the <i>FUS1</i> promoter the cells also go in growth arrest mediated by the <i>FAR1</i> promoter. However it is undesirable that the cells stop growing once they respond to a ligand. Therefore it is needed to knock out the <i>FAR1</i> promoter. Therefore we characterized the knockout of the <i>FAR1</i> promoter. <br/><br />
<h3>Increasing sensitivity</h3><br />
To optimize the signal transduction from the receptor to the downstream cascade, a mammalian alpha subunit can be introduced which has affinity with the RI7-receptor [1]. For this, making a knockout of the native <i>GPA1</i> gene is needed in order to let the subunit work as substitute. In the results we describe how we generated a knockout in yeast. In future work the alpha subunit should be expressed and characterized. </p><br />
<br />
<br />
<a name="P9"> <h2>Parts</h2> </a> <br />
<p>The receptor constructs and the reporter constructs are combined to have one complete olfactory system. The following biobricks are created:</p><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/d/d2/GPR109A_snifferomyces.jpg" width="620" /><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775005 " target="_blank">BBa_K775005</a><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/83/OLF154_snifferomyces.jpg" width="620"/><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775006 " target="_blank">BBa_K775006</a><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/1/16/ORIG1_snifferomyces.jpg" width="620"/><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775007 " target="_blank">BBa_K775007</a><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/3/3f/Odr10_snifferomyces.jpg" width="620"/><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775008 " target="_blank">BBa_K775008</a><br/><br/><br />
<a name="P8"><h2>Results</h2></a><br />
<br />
<h3>Characterization <i>Far1</i> Knockout </h3><br />
<p>The <i>FAR1</i> has been knocked out to prevent growth arrest once a ligand has been added. To test this we have measured the optical density of Wild Type and Knockout strains with and without alpha feromones. <br/><br />
<br/><br />
The results seem to show that there is a reduction in growth in both strains after the addition of alpha-feromones, although the <i>Δfar1</i>-strain shows less reduction. </br><br />
Another remarkable feature is that the <i>Δfar1</i>-strain generally appears to grow faster than the Wild-type strain. A possible explanation is that the gene for growth-arrest has been knocked out. The <i>FAR1</i> is not only related to the mating response, but is also a control in the mitotic cycle of budding yeast. [2] </br><br />
</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/6/66/Groeicurve.jpg" align="middle" width="100%"><br />
<br />
<br />
<h3>Transformations</i></h3><br />
<p>Yeast strains were successfully transformed with the <i>GPR109A</i> receptor and output <br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775005" target="_blank">BBa_K775005</a>, called <b>NR1</b> (nicotinic acid reporter). The <i>RI7-ODR10</i> receptor and output <br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775008" target="_blank">BBa_K775008</a>. <i>Olfr154</i> receptor and output <br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775010" target="_blank">BBa_K775010</a>, called <b>BR1</b> (banana reporter 1) and <i>RI7-OR1G1</i> receptor and output <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775011">BBa_K775011</a>, called <b>BR2</b> (banana reporter 2).</p><br />
<br/><br />
<p><br />
<h3>Niacin Snifferomyces</h3><br/><br />
<h6>Setup</h6><br/><br />
In order to test the yeast NR1 cells with nicotinic acid receptor and the receptor inducible GFP reporter we induced the cells with nicotinic acid or alpha pheromone and measured the cellular response through flow cytometry.<br />
<h6>Outcome</h6><br/><br />
Flow cytometry data of +NR1 cells induced with nicotinic acid or alpha pheromones is shown.<br />
</p><br />
<img src="https://static.igem.org/mediawiki/2012/c/c4/Flow_Cytometry_Snifferomyces_NR1_graph.png" width="350"><br />
<p><br />
Here we see that addition of receptor ligand doesn’t induce a response in comparison with alpha induced cells. Also in terms of a cutoff comparison in terms of percentages this seems to be the case. Possible explanations are:<br />
<ul><li>A too small subset of the yeast receptors is localized in the membrane.</li><br />
<li>The affinity of the receptor with the alpha subunit is too low.</li><br />
<br />
<br />
<br />
<h3>Isoamyl acetate Snifferomyces</h3><br />
<h6>Setup</h6><br />
<h6>Outcome</h6><br />
<br />
<a name="P7"> <h2>Conclusions</h2> </a><br />
<p>Four olfactory system biobricks were added to the registry. </p><br/><br />
<a name="P9"><h2>References</h2> </a><br />
<h6>[1] Jasmina Minic, Marie-annick Persuy, Elodie Godel, Josiane Aioun, Ian Connerton, Roland Salesse, Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening, FEBS Journal (2005)</h6><br />
<h6>[2] "A cell sizer network involving Cln3 and Far1 controls entrance into S phase in the mitotic cycle of budding yeast" Alberghina et al. November 1, 2004 // JCB vol. 167 no. 3 433-443 </h6><br />
<br />
</div><br />
<img src="https://static.igem.org/mediawiki/igem.org/3/37/Footer_2.jpg" align="middle" width="690"><br />
<a href='https://2012.igem.org/Main_Page' target="_blank"><div id='logo_igem2'></div><a/></body></html></div>MarkWeijershttp://2012.igem.org/Team:TU-Delft/part3Team:TU-Delft/part32012-10-26T22:31:43Z<p>MarkWeijers: </p>
<hr />
<div>{{:Team:TUDelft/Scripts}}{{:Team:TUDelft/CSS}}{{:Team:TUDelft/menu}}<br />
<html><br />
<head><title>Receptor</title></head><br />
<body><br />
<div style="height:70px; width:100%;"></div><br />
<div id="logo_ed"><a href="https://2012.igem.org/Team:TU-Delft" 'onfocus=this.blur()'><img src="https://static.igem.org/mediawiki/igem.org/8/88/Logoigemklein.png" border="0" width="100" height="100"></a></div><br />
<br />
<div id="contentbox" style="text-align:justify;"><br />
<img src="https://static.igem.org/mediawiki/igem.org/e/e4/Snifferomyces.jpg" align="middle" width="100%"><br />
<br />
<br />
<h2>Content</h2> <br />
<a href="#P10"> Introduction</a><br><br />
<a href="#P9">Parts </a><br><br />
<a href="#P8">Results </a><br><br />
<a href="#P7"> Conclusions</a><br><br />
<a href="#P9">References</a><br><br />
<br />
<a name="P10"> <br><h2>Introduction</h2> </a><br />
<h3>Yeast with olfactory receptor+reporter=Snifferomyces</h3><br />
<p>By combining the olfactory receptor and the <i>FUS1pr-EGFP</i> reporter, a complete yeast olfactory system is obtained. If the corresponding ligand binds to the receptor the <i>FAR1</i> promoter is turned on and the EGFP is expressed. This EGFP signal can be read out by a fluorescence meter. If the olfactory system will be implemented as a diagnostics tool in developing countries, the EGFP reporter should be changed by a visible reporter.<br/><br />
<h3>Growth arrest</h3><br />
Besides the induction of the <i>FUS1</i> promoter the cells also go in growth arrest mediated by the <i>FAR1</i> promoter. However it is undesirable that the cells stop growing once they respond to a ligand. Therefore it is needed to knock out the <i>FAR1</i> promoter. Therefore we characterized the knockout of the <i>FAR1</i> promoter. <br/><br />
<h3>Increasing sensitivity</h3><br />
To optimize the signal transduction from the receptor to the downstream cascade, a mammalian alpha subunit can be introduced which has affinity with the RI7-receptor [1]. For this, making a knockout of the native <i>GPA1</i> gene is needed in order to let the subunit work as substitute. In the results we describe how we generated a knockout in yeast. In future work the alpha subunit should be expressed and characterized. </p><br />
<br />
<br />
<a name="P9"> <h2>Parts</h2> </a> <br />
<p>The receptor constructs and the reporter constructs are combined to have one complete olfactory system. The following biobricks are created:</p><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/d/d2/GPR109A_snifferomyces.jpg" width="620" /><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775005 " target="_blank">BBa_K775005</a><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/83/OLF154_snifferomyces.jpg" width="620"/><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775006 " target="_blank">BBa_K775006</a><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/1/16/ORIG1_snifferomyces.jpg" width="620"/><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775007 " target="_blank">BBa_K775007</a><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/3/3f/Odr10_snifferomyces.jpg" width="620"/><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775008 " target="_blank">BBa_K775008</a><br/><br/><br />
<a name="P8"><h2>Results</h2></a><br />
<br />
<h3>Characterization <i>Far1</i> Knockout </h3><br />
<p>The <i>FAR1</i> has been knocked out to prevent growth arrest once a ligand has been added. To test this we have measured the optical density of Wild Type and Knockout strains with and without alpha feromones. <br/><br />
<br/><br />
The results seem to show that there is a reduction in growth in both strains after the addition of alpha-feromones, although the <i>Δfar1</i>-strain shows less reduction. </br><br />
Another remarkable feature is that the <i>Δfar1</i>-strain generally appears to grow faster than the Wild-type strain. A possible explanation is that the gene for growth-arrest has been knocked out. The <i>FAR1</i> is not only related to the mating response, but is also a control in the mitotic cycle of budding yeast. [2] </br><br />
</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/6/66/Groeicurve.jpg" align="middle" width="100%"><br />
<br />
<br />
<h3>Transformations</i></h3><br />
<p>Yeast strains were successfully transformed with the <i>GPR109A</i> receptor and output <br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775005" target="_blank">BBa_K775005</a>, called <b>NR1</b> (nicotinic acid reporter). The <i>RI7-ODR10</i> receptor and output <br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775008" target="_blank">BBa_K775008</a>. <i>Olfr154</i> receptor and output <br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775010" target="_blank">BBa_K775010</a>, called <b>BR1</b> (banana reporter 1) and <i>RI7-OR1G1</i> receptor and output <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775011">BBa_K775011</a>, called <b>BR2</b> (banana reporter 2).</p><br />
<br/><br />
<p><br />
<h3>Niacin Snifferomyces</h3><br/><br />
<h6>Setup</h6><br/><br />
In order to test the yeast NR1 cells with nicotinic acid receptor and the receptor inducible GFP reporter we induced the cells with nicotinic acid or alpha pheromone and measured the cellular response through flow cytometry.<br />
<h6>Outcome</h6><br/><br />
Flow cytometry data of +NR1 cells induced with nicotinic acid or alpha pheromones is shown.<br />
</p><br />
<img src=”https://static.igem.org/mediawiki/2012/c/c4/Flow_Cytometry_Snifferomyces_NR1_graph.png” width=”350”><br />
<p><br />
Here we see that addition of receptor ligand doesn’t induce a response in comparison with alpha induced cells. Also in terms of a cutoff comparison in terms of percentages this seems to be the case. Possible explanations are:<br />
<ul><li>A too small subset of the yeast receptors is localized in the membrane.</li><br />
<li>The affinity of the receptor with the alpha subunit is too low.</li><br />
<br />
<br />
<br />
<h3>Isoamyl acetate Snifferomyces</h3><br />
<h6>Setup</h6><br />
<h6>Outcome</h6><br />
<br />
<a name="P7"> <h2>Conclusions</h2> </a><br />
<p>Four olfactory system biobricks were added to the registry. </p><br/><br />
<a name="P9"><h2>References</h2> </a><br />
<h6>[1] Jasmina Minic, Marie-annick Persuy, Elodie Godel, Josiane Aioun, Ian Connerton, Roland Salesse, Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening, FEBS Journal (2005)</h6><br />
<h6>[2] "A cell sizer network involving Cln3 and Far1 controls entrance into S phase in the mitotic cycle of budding yeast" Alberghina et al. November 1, 2004 // JCB vol. 167 no. 3 433-443 </h6><br />
<br />
</div><br />
<img src="https://static.igem.org/mediawiki/igem.org/3/37/Footer_2.jpg" align="middle" width="690"><br />
<a href='https://2012.igem.org/Main_Page' target="_blank"><div id='logo_igem2'></div><a/></body></html></div>MarkWeijershttp://2012.igem.org/Team:TU-Delft/part3Team:TU-Delft/part32012-10-26T22:30:02Z<p>MarkWeijers: </p>
<hr />
<div>{{:Team:TUDelft/Scripts}}{{:Team:TUDelft/CSS}}{{:Team:TUDelft/menu}}<br />
<html><br />
<head><title>Receptor</title></head><br />
<body><br />
<div style="height:70px; width:100%;"></div><br />
<div id="logo_ed"><a href="https://2012.igem.org/Team:TU-Delft" 'onfocus=this.blur()'><img src="https://static.igem.org/mediawiki/igem.org/8/88/Logoigemklein.png" border="0" width="100" height="100"></a></div><br />
<br />
<div id="contentbox" style="text-align:justify;"><br />
<img src="https://static.igem.org/mediawiki/igem.org/e/e4/Snifferomyces.jpg" align="middle" width="100%"><br />
<br />
<br />
<h2>Content</h2> <br />
<a href="#P10"> Introduction</a><br><br />
<a href="#P9">Parts </a><br><br />
<a href="#P8">Results </a><br><br />
<a href="#P7"> Conclusions</a><br><br />
<a href="#P9">References</a><br><br />
<br />
<a name="P10"> <br><h2>Introduction</h2> </a><br />
<h3>Yeast with olfactory receptor+reporter=Snifferomyces</h3><br />
<p>By combining the olfactory receptor and the <i>FUS1pr-EGFP</i> reporter, a complete yeast olfactory system is obtained. If the corresponding ligand binds to the receptor the <i>FAR1</i> promoter is turned on and the EGFP is expressed. This EGFP signal can be read out by a fluorescence meter. If the olfactory system will be implemented as a diagnostics tool in developing countries, the EGFP reporter should be changed by a visible reporter.<br/><br />
<h3>Growth arrest</h3><br />
Besides the induction of the <i>FUS1</i> promoter the cells also go in growth arrest mediated by the <i>FAR1</i> promoter. However it is undesirable that the cells stop growing once they respond to a ligand. Therefore it is needed to knock out the <i>FAR1</i> promoter. Therefore we characterized the knockout of the <i>FAR1</i> promoter. <br/><br />
<h3>Increasing sensitivity</h3><br />
To optimize the signal transduction from the receptor to the downstream cascade, a mammalian alpha subunit can be introduced which has affinity with the RI7-receptor [1]. For this, making a knockout of the native <i>GPA1</i> gene is needed in order to let the subunit work as substitute. In the results we describe how we generated a knockout in yeast. In future work the alpha subunit should be expressed and characterized. </p><br />
<br />
<br />
<a name="P9"> <h2>Parts</h2> </a> <br />
<p>The receptor constructs and the reporter constructs are combined to have one complete olfactory system. The following biobricks are created:</p><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/d/d2/GPR109A_snifferomyces.jpg" width="620" /><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775005 " target="_blank">BBa_K775005</a><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/8/83/OLF154_snifferomyces.jpg" width="620"/><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775006 " target="_blank">BBa_K775006</a><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/1/16/ORIG1_snifferomyces.jpg" width="620"/><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775007 " target="_blank">BBa_K775007</a><br/><br />
<img src="https://static.igem.org/mediawiki/igem.org/3/3f/Odr10_snifferomyces.jpg" width="620"/><br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775008 " target="_blank">BBa_K775008</a><br/><br/><br />
<a name="P8"><h2>Results</h2></a><br />
<br />
<h3>Characterization <i>Far1</i> Knockout </h3><br />
<p>The <i>FAR1</i> has been knocked out to prevent growth arrest once a ligand has been added. To test this we have measured the optical density of Wild Type and Knockout strains with and without alpha feromones. <br/><br />
<br/><br />
The results seem to show that there is a reduction in growth in both strains after the addition of alpha-feromones, although the <i>Δfar1</i>-strain shows less reduction. </br><br />
Another remarkable feature is that the <i>Δfar1</i>-strain generally appears to grow faster than the Wild-type strain. A possible explanation is that the gene for growth-arrest has been knocked out. The <i>FAR1</i> is not only related to the mating response, but is also a control in the mitotic cycle of budding yeast. [2] </br><br />
</p><br />
<img src="https://static.igem.org/mediawiki/igem.org/6/66/Groeicurve.jpg" align="middle" width="100%"><br />
<br />
<br />
<h3>Transformations</i></h3><br />
<p>Yeast strains were successfully transformed with the <i>GPR109A</i> receptor and output <br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775005" target="_blank">BBa_K775005</a>, called <b>NR1</b> (nicotinic acid reporter). The <i>RI7-ODR10</i> receptor and output <br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775008" target="_blank">BBa_K775008</a>. <i>Olfr154</i> receptor and output <br />
<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775010" target="_blank">BBa_K775010</a>, called <b>BR1</b> (banana reporter 1) and <i>RI7-OR1G1</i> receptor and output <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K775011">BBa_K775011</a>, called <b>BR2</b> (banana reporter 2).</p><br />
<br/><br />
<p><br />
<h3>Niacin Snifferomyces</h3><br/><br />
<h6>Setup</h6><br/><br />
In order to test the yeast NR1 cells with nicotinic acid receptor and the receptor inducible GFP reporter we induced the cells with nicotinic acid or alpha pheromone and measured the cellular response through flow cytometry.<br />
<h6>Outcome</h6><br/><br />
Flow cytometry data of +NR1 cells induced with nicotinic acid or alpha pheromones is shown.<br />
</p><br />
<img src=”https://static.igem.org/mediawiki/2012/c/c4/Flow_Cytometry_Snifferomyces_NR1_graph.png” alt="FC results" width=”350”><br />
<p><br />
Here we see that addition of receptor ligand doesn’t induce a response in comparison with alpha induced cells. Also in terms of a cutoff comparison in terms of percentages this seems to be the case. Possible explanations are:<br />
<ul><li>A too small subset of the yeast receptors is localized in the membrane.</li><br />
<li>The affinity of the receptor with the alpha subunit is too low.</li><br />
<br />
<br />
<br />
<h3>Isoamyl acetate Snifferomyces</h3><br />
<h6>Setup</h6><br />
<h6>Outcome</h6><br />
<br />
<a name="P7"> <h2>Conclusions</h2> </a><br />
<p>Four olfactory system biobricks were added to the registry. </p><br/><br />
<a name="P9"><h2>References</h2> </a><br />
<h6>[1] Jasmina Minic, Marie-annick Persuy, Elodie Godel, Josiane Aioun, Ian Connerton, Roland Salesse, Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening, FEBS Journal (2005)</h6><br />
<h6>[2] "A cell sizer network involving Cln3 and Far1 controls entrance into S phase in the mitotic cycle of budding yeast" Alberghina et al. November 1, 2004 // JCB vol. 167 no. 3 433-443 </h6><br />
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</div><br />
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