Team:TecMonterrey/methods.html

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         </div>
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<div id="dude">
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    <div id="grid">
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        <h1>Methods</h1>
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            <div id="fondo">
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        <div style="position:relative"><a class="ancla" name="GNL"></a></div>
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                <div></div>
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        <h2>General Protocols</h2>
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                <h2 id="boton">return</h2>
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            </div>
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<h3>Transformation and Confirmation</h3>
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            <div id="101" class="cell" style="left:8px">
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                <h3 id="1" class="mes">week 1</h3>
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<p>The <i>Escherichia coli</i> strains used for our experiments were <i>TOP10</i> and <i>BL21 (DE3)</i>. DNA expression cassettes were confirmed by restriction enzyme digestion with EcoRI-HF (NEB) and agarose electrophoresis.</p>  
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                <p id="a" class="mes">FEB 27</p>
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            </div>
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<h3>Induction</h3>
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            <div id="102" class="cell" style="left:172px">
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                <h3 id="1" class="mes">week 2</h3>
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<p>50 mL tubes were inoculated with each of the transformed <i>BL21 (DE3)</i> strains and incubated overnight in LB medium with the proper antibiotic (100ug/mL of ampicillin, 50 µg/mL of kanamycin, 35 µg/mL of chloramphenicol or 15 µg/mL of tetracycline) at 37&#176;C and 250 rpm. After 12 hours of incubation, 250 mL flasks with 50 mL of LB medium with antibiotic were inoculated until 0.1 O.D.600nm (Genesys 10UV scanning, Thermo scientific) with the proper strain. The flasks were then incubated at 37&#176;C and 250 rpm until they reached to an O.D.600nm between 0.4 and 0.6.</p>
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                <p id="b" class="mes">APR 1 - APR 7</p>
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            </div>
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<p>Cells were pelleted (IEC CL40R centrifuge, Thermo scientific) at 4&#176;C and 4,000 g for 10 minutes, the medium was discarded and 50 mL of fresh medium were added and supplemented antibiotic and 0.1 mM IPTG and/or 1% L-arabinose. Cells were then incubated at 27&#176;C and 250 rpm for 8 hours.</p>
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            <div id="103" class="cell" style="left:336px">
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                <h3 id="1" class="mes">week 3</h3>
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<h3>Cell Rupture</h3>
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                <p id="c" class="mes">MAY 26 - MAY 30</p>
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            </div>
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<p>Cells were harvested by centrifugation at 4&#176;C and 5,000 g during 10 minutes. Supernatant was discarded and the pellets were resuspended in 7 mL per gram of pellet in lysis buffer (5% v/v Tween-20 in 25 mM K2HPO4 pH 7.5).</p>  
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            <div id="104" class="cell" style="right:336px">
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                <h3 id="1" class="mes">week 4</h3>
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<p>Cells were sonicated (Branson Sonifier 150) on ice, during 3.17 minutes with 10 seconds intervals (10 seconds pulses and 10 seconds for cooling). The lysate was then centrifuged at 4&#176;C for 15 minutes at 5,000 g.</p>
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                <p id="d" class="mes">JUN 5 - JUN 7</p>
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            </div>
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<p>The soluble fractions were recovered, while the insoluble fractions were suspended with 5 mL lysis buffer pH 7.2 supplemented with 8 M urea, incubated at 37&#176;C for 1 hour and then centrifuged at 4&#176;C and 10,000 g for 20 minutes. The supernatant containing the solubilized fraction was recovered. Both the soluble and insoluble-treated fractions were measured for protein concentration by Biuret assay (200µL of sample + 800µL of Biuret reagent incubated for 15 minutes at room temperature).</p>
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            <div id="105" class="cell" style="right:172px">
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                <h3 id="1" class="mes">week 5</h3>
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<h3>Purification</h3>
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                <p id="e" class="mes">JUN 10 - JUN 13</p>
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            </div>
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<p>Purification was conducted according to the <a class="t" href="http://www.piercenet.com/instructions/2162206.pdf">HisPur Ni-NTA Spin Purification Kit recommendations</a> (Thermo Scientific, product number #88229).</p>
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            <div id="106" class="cell" style="right:8px">
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                <h3 id="1" class="mes">week 6</h3>
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<h3>Concentration</h3>
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                <p id="f" class="mes">JUN 17 - JUN 21</p>
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            </div>
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<p>The eluents from the purification (around 9 ml) were concentrated with 3 KDa ultrafiltration tubes until only 1.5 ml remained. (Amicon Ultra-15 Centrifugal Filter Units, Catalogue number #UFC900324). After concentration, protein quantity was determined by Biuret's method.</p>  
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            <div id="107" class="cell" style="left:8px;top:154px">
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                <h3 id="1" class="mes">week 7</h3>
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<h3>Tricine-SDS PAGE</h3>
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                <p id="g" class="mes">JUN 24 - JUN 28</p>
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            </div>
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<p>The Tricine-SDS PAGE was done accordingly to the protocol by (Schägger, H., 2006), on a Miniprotean 3 cell (BIO-RAD).</p>
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            <div id="108" class="cell" style="left:172px;top:154px">
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                <h3 id="1" class="mes">week 8</h3>
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<h3>Western Blot</h3>
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                <p id="h" class="mes">JUL 8 - JUL 11</p>
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            </div>
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<p>The Tricine-SDS PAGE gel was then blotted into a PVDF membrane according to the protocol mentioned in the “Mini Trans Blot" manual (BIO-RAD) and the suggested transfer buffer (25 mM Tris, pH 8.3, 192 mM glycine) during 1 hour at 100 Volts.
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            <div id="109" class="cell" style="left:336px;top:154px">
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The Western blot was done accordingly to the <a class="t" href="http://www.piercenet.com/instructions/2162133.pdf">Pierce&#174; Fast Western Blot Kit, ECL Substrate protocol</a> (Thermo Scientific Catalogue Number #35050). Using as primary antibody an Anti-polyHistidine Peroxidase Conjugated antibody (Sigma Catalog number #A7058).</p>
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                <h3 id="1" class="mes">week 9</h3>
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<div style="position:relative"><a class="ancla" name="THPR"></a></div>
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                <p id="i" class="mes">JUL 15 - JUL 22</p>
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<h2>Therapeutic Proteins Methods</h2>
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            </div>
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            <div id="110" class="cell" style="right:336px;top:154px">
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<h3>Transformation and Confirmation</h3>
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                <h3 id="1" class="mes">week 10</h3>
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                <p id="j" class="mes">JUL 31 - AUG 3</p>
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<p>The transformation and confirmation protocol were followed as established in the <i>General protocols</i> section. The strains were transformed with the expression cassettes: </p>
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            </div>
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            <div id="111" class="cell" style="right:172px;top:154px">
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<h4 class="BioI">Tat-Apoptin</h4>
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                <h3 id="1" class="mes">week 11</h3>
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                <p id="k" class="mes">AUG 5 - AUG 7</p>
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<img class="BB" src="https://static.igem.org/mediawiki/igem.org/0/09/DESCRIPTION5.png">
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            </div>
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            <div id="112" class="cell" style="right:8px;top:154px">
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<h4 class="BioI">TRAIL</h4>
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                <h3 id="1" class="mes">week 12</h3>
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                <p id="l" class="mes">AUG 12 - AUG 17</p>
-
<img class="BB" src="https://static.igem.org/mediawiki/igem.org/f/f8/DESCRIPTION6.png">
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            </div>
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            <div id="113" class="cell" style="left:8px;bottom:0px">
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<h4 class="BioI">Tat-Apoptin-HlyA</h4>
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                <h3 id="1" class="mes">week 13</h3>
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                <p id="m" class="mes">AUG 21 - AUG 22</p>
-
<img class="BB" src="https://static.igem.org/mediawiki/igem.org/c/c6/DESCRIPTION3.png">
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            </div>
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            <div id="114" class="cell" style="left:172px;bottom:0px">
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<h4 class="BioI">TRAIL-HlyA</h4>
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                <h3 id="1" class="mes">week 14</h3>
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                <p id="n" class="mes">AUG 26 - AUG 28</p>
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<img class="BB" src="https://static.igem.org/mediawiki/igem.org/c/cc/DESCRIPTION4.png">
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            </div>
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            <div id="115" class="cell" style="left:336px;bottom:0px">
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                <h3 id="1" class="mes">week 15</h3>
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<p>The transformations were confirmed by selective growth of colonies in plates with Ampicillin (100ug/ml) Furthermore the colonies were confirmed by restriction enzyme digestion with EcoRI-HF (NEB) and electrophoresis. </p>
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                <p id="o" class="mes">SEP 2 - SEP 8</p>
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            </div>
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<h3>Induction, Cell Rupture, Purification, Concentration, Tricine-SDS PAGE and Western Blot</h3>
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            <div id="116" class="cell" style="right:336px;bottom:0px">
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                <h3 id="1" class="mes">week 16</h3>
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<p>The induction, cell rupture, purification, concentration, Tricine-SDS PAGE and Western Blot protocols were performed as specified in the General protocols section with no modifications.</p>
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                <p id="p" class="mes">SEP 12 - SEPT 15</p>
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            </div>
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<h3>CitotoxicityMTT Assay</h3>
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            <div id="117" class="cell" style="right:172px;bottom:0px">
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                <h3 id="1" class="mes">week 17</h3>
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<p>The MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromid, is based on the conversion of the reagent MTT into formazan crystals by viable mammal cells, due to the activity of a specific enzyme located in the mitochondrion.  By exploiting this enzymatic activity of dehydrogenase enzymes, the assay can indirectly measure the cell viability of a cell culture through its metabolic activity. This method is one of the most used to measure cytotoxic activity of drugs, extracts and compounds on different cell lines (Meerloo, J. v., Kaspers, G. J., & Cloos, J., 2011).
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                <p id="o" class="mes">SEP 16 - SEP 21</p>
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            </div>
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<p>The assay conditions used in the experiment may influence the results and must be considered during the analysis of data. The age of cell lines, the passage number and requisites of the medium may be important factors that can affect the results.  Due to variation on the requirements and growth rates of each cell line, it is difficult to establish specific guidelines for the execution of this experiment; however, cells are usually cultivated at a cell density of 5,000-10,000 cells per well (this density should be enough to reach an optimum density in 48-72 hours).  The presence of phenol red may affect seriously the result, and for this reason it is highly recommended to culture the cells in a phenol red free medium (Vybrant® MTT Cell Proliferation Assay Kit, 2002).</p>
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            <div id="118" class="cell" style="right:8px;bottom:0px">
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                <h3 id="1" class="mes">week 18</h3>
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<p>The assay is divided in different procedures:</p>
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                <p id="p" class="mes">SEP 22 - SEPT 26</p>
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<ol>
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            </div>
-
<li>96 well cell culture plate preparation.</li>
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        </div>
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<li>Sample addition to 96 well cell culture plate.</li>
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-
<li>Cell viability measurement.</li>
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-
<li>Data analysis.</li>
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</ol>
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<ol class="letras">
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<li><b>96 well cell culture plate preparation.</b><br>
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<i>During this first part of the assay, cells are transferred from a culture dish to a 96 well cell culture plate.</i></li>
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    <br>
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<ol>
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<li>Exponentially grown cells were trypsinized according to (Trypsinization of adherent cells, 2002).</li>
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<li>The cells were suspended in D-MEM/10% SBF medium until it reached a cell density of 5x105 cells/ml.</li>
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<li>For each well, 100µl were plated (50,000 cells) on a 96 well microplate according to the layout shown in Appendix 1.</li>
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-
<li>The cells were incubated for 24 hours at 37oC and 5% C02.</li>
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-
</ol>
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    <br>
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-
<li><b>Sample addition to culture plate.</b><br>
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<i>After 24 hours of incubation of the cell culture microplate, the sample (extract or isolated compound) to be evaluated is added to the culture plate.</i></li>
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    <br>
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-
<ol>
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-
<li>Solutions of the samples (TRAIL and Tat-Apoptin) at different concentrations were prepared by diluting in D-MEM/10%SBF medium.<br>
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-
* For each concentration, prepare 450 µl.</li>
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-
<li>The medium from the microplate was discarded and the cells were washed twice with PBS pH 7.4</li>
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-
<li>Samples were added to the wells according to the layout shown in Appendix 1.<br>
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-
* For the sample control and blank wells, only was added 100 µl of D-MEM/10%SBF medium.</li>
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-
<li>Cell cultures were homogenized with soft “eight shaped movements" on the surface of the hood.</li>
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-
<li>The microplate was incubated for 48 hours at 37 oC and 5% C02.</li>
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-
</ol>
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-
    <br>
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-
<li><b>Cell viability measurement.</b><br>
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<i>After 48 hours of incubation with the extract or compound to be evaluated, cell viability is measured with the MTT reagent.</i></li>
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    <br>
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-
<ol>
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<li>20 µL of MTT reagent were added to each one of the 96 wells.</li>
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-
<li>Culture plates were placed back into the culture chamber at 37oC and 5% CO2 for 1 hour.</li>
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-
<li>Absorbance was read at 570 nm.<br>
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-
*To ensure that the protocol was performed correctly, make sure that the wells have an absorbance between 0.9&#8249;Abs&#8249;1.</li>
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-
<li>Cell culture medium and culture plate were disposed of in the proper containers.</li>
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-
</ol>
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-
<br>
+
-
<li><b>Data analysis</b></li>
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-
<ol>
+
-
<li>
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-
<p>For each sample concentration, the following calculation was made:</p>
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+
-
<img src="https://static.igem.org/mediawiki/2013/2/22/Ecua1.png">
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<p>Where:</p>
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<img src="https://static.igem.org/mediawiki/2013/3/31/Ecua2.png">
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<p>n= 3<br>
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a= Sample concentration replicates</p>
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<img src="https://static.igem.org/mediawiki/2013/8/8e/Ecua3.png">
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<p>m= 18<br>
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b= Cells replicates</p>
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<img src="https://static.igem.org/mediawiki/2013/b/bf/Ecua4.png">
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<p>z= <br>
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c= Blank replicates</p>
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</li>
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-
<li>Once the cell viability was obtained for each concentration, the data was plotted by comparing concentration vs. cell viability.<br>
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-
      *The adjustment of the data can be made with different regression models: linear, quadratic, exponential, etc. depending on the data obtained.</li>
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<li>With a regression model, the concentration of sample for which it is reached 50% of cell viability (IC50) was obtained. </li>
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-
</ol>
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</ol>
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<div style="position:relative"><a class="ancla" name="SCRT"></a></div>
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<h2>Secretion System Methods</h2>
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<p>For the secretion module, our team took two different approaches. The first one made use of <i>Escherichia coli</i> BL21 (DE3) co-transformed with a GFP fused to a HlyA secretion peptide in its C terminus and another construct containing the proteins needed for secretion HlyB and HlyD; as a negative control another <i>E. coli</i> BL2 (DE3) was co-transformed with a non-tagged GFP and the same construct containing the proteins needed for secretion HlyB and HlyD in order to measure and compare fluorescence from the contents outside the cell, and those inside (Supernatant and soluble fractions).  For a second approach we co-transformed <em>E. coli BL21</em> (DE3) with a construct containing TAT-Apoptin tagged with both a Histidine6x tag and the secretion signal HlyA with another construct containing the proteins needed for secretion HlyB and HlyD; then we con-transformed <i>E. coli</i> BL21 (DE3) with a construct containing TRAIL tagged with both a Histidine6x tag and the secretion signal HlyA with the same construct containing HlyB and HlyD. Finally we did a Western Blot assay in order to determine and compare presence of TAT-Apoptin-HlyA and TRAIL-HlyA in the supernatant and soluble fractions of the cell cultures.</p>
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<h3>Co-Transformation and Confirmation</h3>
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<p>The <i>E. coli</i> strains (TOP10 or BL21 (DE3)) previously transformed with the construct containing HlyB and HlyD were re-transformed with GFP-HlyA, TAT-Apoptin-HlyA and TRAIL-HlyA, producing the following strains:</p>
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<h4 class="BioI">GFP-HlyA+Secretion System</h4>
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-
 
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-
<img class="BB" src="https://static.igem.org/mediawiki/igem.org/8/8a/DESCRIPTION12.png">
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<img class="BB" src="https://static.igem.org/mediawiki/igem.org/3/3f/DESCRIPTION14.png">
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<h4 class="BioI">GFP+Secretion System</h4>
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<img class="BB" src="https://static.igem.org/mediawiki/igem.org/9/9a/DESCRIPTION13.png">
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<img class="BB" src="https://static.igem.org/mediawiki/igem.org/3/3f/DESCRIPTION14.png">
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<h4 class="BioI">TRAIL-HlyA+Secretion System</h4>
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+
-
<img class="BB" src="https://static.igem.org/mediawiki/igem.org/c/cc/DESCRIPTION4.png">
+
-
 
+
-
<img class="BB" src="https://static.igem.org/mediawiki/igem.org/3/3f/DESCRIPTION14.png">
+
-
 
+
-
<h4 class="BioI">TAT-Apoptin-HlyA+Secretion System</h4>
+
-
 
+
-
<img class="BB" src="https://static.igem.org/mediawiki/igem.org/c/c6/DESCRIPTION3.png">
+
-
 
+
-
<img class="BB" src="https://static.igem.org/mediawiki/igem.org/3/3f/DESCRIPTION14.png">
+
-
 
+
-
<p>The transformations were confirmed by selective growth of colonies in plates with both Kanamycin (50ug/ml) and Ampicillin (100ug/ml) since the secretion construct contain a Kanamycin resistance gene and the GFP, TRAIL-HlyA and TAT-Apoptin-HlyA containg an ampicillin resistance gene. Furthermore the colonies were confirmed by restriction enzyme digestion with EcoRI-HF (NEB) and electrophoresis.</p>
+
-
 
+
-
<h3>Induction</h3>
+
-
 
+
-
<p>50 mL tubes were inoculated with each of the transformed <i>BL21 (DE3)</i> strains and incubated overnight in LB medium with the proper antibiotic (100ug/mL of ampicillin and 50 µg/mL of kanamycinat) 37&#176;C and 250 rpm. After 12 hours of incubation, 250 mL flasks with 50 mL of LB medium and antibiotic were inoculated until 0.1 O.D.600nm (Genesys 10UV scanning, Thermo scientific) with the proper strain. The flasks were then incubated at 37&#176;C and 250 rpm until they reached to an O.D.600nm 1.</p>
+
-
 
+
-
<p>Cells were pelleted (IEC CL40R centrifuge, Thermo scientific) at 4&#176;C and 4,000 g for 10 minutes, the medium was discarded and 50 mL of fresh medium were added and supplemented with the proper antibiotic, 0.1 mM IPTG and 1% L-arabinose. Cells were then incubated at 27&#176;C and 250 rpm for 8 hours.</p>
+
-
 
+
-
<h3>Fluorescence Measurement</h3>
+
-
 
+
-
<p>In the case of GFP and GFP-HlyA, supernatants and soluble fractions were analyzed for fluorescence in a Biotek Synergy HT, plate reader (Excitation 485/20, Emission 528/20, Sensitivity 20).</p>
+
-
 
+
-
<h3>Cell Rupture</h3>
+
-
 
+
-
<p>Cell rupture was performed as specified in the General protocols section with no modifications. The soluble fraction from the lysate and the supernatant from induction were kept at 4 &#176;C.</p>
+
-
 
+
-
<h3>Purification, Concentration</h3>
+
-
 
+
-
<p>Purification was only required for the proteins with a Histidine6x tag, TRAIL-HlyA, TAT-Apoptin-HlyA, and both the supernatant and the soluble fractions were purified as specified in the <i>General Protocols</i> section.</p>
+
-
 
+
-
<h3>Tricine-SDS-Page</h3>
+
-
 
+
-
<p>Tricine-SDS PAGE was performed as specified in the <i>General Protocols</i> section with no modifications.</p>
+
-
 
+
-
<h3>Western Blot</h3>
+
-
 
+
-
<p>Only those proteins with a histidine6x tag were blotted as specified in the <i>General Protocols</i> section with no modifications.</p>
+
-
 
+
-
<div style="position:relative"><a class="ancla" name="INTL"></a></div>
+
-
<h2>Internalization of TAT-GFP Protein into Eukaryotic Cells</h2>
+
-
 
+
-
<p>This experiment aims to prove that the Tat peptide, a protein transduction domain (PDT) from the human immunodeficiency virus (HIV), is capable of deliver proteins into eukaryotic cells. To achieve this, the carrier Tat peptide and the transported protein (green fluorescent protein, GFP) were associated by genetic construction leading to a fusion protein expressing the PTD at its N-terminus followed by a His-Tag sequence. Taking advantage of the characteristics of the GFP, the protocol described by (Silhol, M. et al, 2002) was followed with some modifications.</p>
+
-
<h3>Transformation and Confirmation</h3>
+
-
 
+
-
<p>The transformation and confirmation protocol were followed as established in the <i>General Protocols</i> section. The strains were transformed with the expression cassettes:</p>
+
-
<ul class="texto">
+
-
<li>HG: His GFP</li>
+
-
<li>TG: Tat-GFP</li>
+
-
</ul>
+
-
<h3>Induction, Cell Rupture, Purification and Concentration</h3>
+
-
<p>The induction, cell rupture, purification and concentration protocols were performed as specified in the General protocols section with no modifications.</p>
+
-
<h3>Internalization Assay</h3>
+
-
<ol>
+
-
<li>Exponentially growing cells (NIH-T3 and CACO-2 cell lines) were plated on 24-well microplate (5x105 cells per well) and cultured overnight at 37&#176;C.
+
-
*Three wells were left without cells to use as blank.</li>
+
-
<br>
+
-
<li>The medium was discarded and the cells were washed twice with PBS pH 7.4</li>
+
-
<br>
+
-
<li>The cells were incubated 1 hour at 37&#176;C with 500 µL of 1-10µg/mL of Tat-GFP dissolved in serum-free medium according to the following layout. <br>
+
-
*In the control group we used 1µg/mL GFP alone dissolved in serum-free medium. <br>
+
-
<img src="https://static.igem.org/mediawiki/igem.org/c/ce/Metodo13.png"><br>
+
-
    *CL=cell line, both cell lines used in this experiment were plated as showed above.</li>
+
-
    <br>
+
-
<li>After incubation, medium was removed and stored for further analysis, and the cells were washed three times with PBS pH 7.4</li>
+
-
<br>
+
-
<li>Cells were trypsinized according to (Trypsinization of adherent cells, 2002) and pelleted by centrifugation at 14,000 g for 10 minutes. </li>
+
-
<br>
+
-
<li>Medium was discarded by decantation and the cells were suspended in 100µL of lysis buffer (1% v/v Triton-X100 in 10mM KCl) and incubated in ice for 1 hour with a gently shake every 15 minutes. </li>
+
-
<br>
+
-
<li>Protein concentration was measured by Biuret method (200uL of sample + 800uL of Biuret reagent incubated for 15 minutes at room temperature).</li>
+
-
<br>
+
-
<li>Analyze cell lysates by Tricine-SDS PAGE and Western blot (specific for His-tagged proteins) according to the protocol specified in the General protocols section. </li>
+
-
    <br>
+
-
</ol>
+
-
<p>With the supernatant stored from the fifth step, a second test was performed to quantify the amount of Tat-GFP protein that enters in each cell. The samples were plated in a 96-well microplate according to the following layout:</p>
+
-
<img src="https://static.igem.org/mediawiki/igem.org/0/08/Metodo14.png">
+
-
<p>The microplate was read in the Synergy HT KC4 BioTek fluorescent microplate reader. The emission wavelength was set to 525nm and the excitation wavelength to 485 nm.</p>
+
-
 
+
-
<h3>Internalization Assay. Version II</h3>
+
-
<ol>
+
-
    <li>A 21mL culture of CHO-S cells (7.86x105 cells/mL) was divided into three 15mL tubes.</li><br>
+
-
    <li>The cells were pelleted by centrifugation at 5,000 rpm for 5 minutes.</li><br>
+
-
    <li>The medium was discarded and the cells were washed twice with PBS pH 7.4</li><br>
+
-
    <li>Cells were incubated for 1 hour at 37&#176;C with:</li><br>
+
-
    <ul>
+
-
        <li>Tube 1) 1mL of 1.3 &#181;g / mL of TG dissolved in serum free medium.</li>
+
-
        <li>Tube 2) 1 ml of 1.3 &#181;g / mL of HG dissolved in serum free medium.</li>
+
-
        <li>Tube 3) 1 ml of serum-free medium (control).</li>
+
-
    </ul><br>
+
-
    <li>After incubation, the cells were pelleted by centrifugation at 5,000 rpm for 5 minutes and the medium was removed and stored for further analysis. The cells were washed three times with PBS pH 7.4</li><br>
+
-
    <li>The cells were lysed using the All Prep&#174; DNA/RNA/Protein mini kit.</li><br>
+
-
    <li>The cell lysates and the supernatants were analyzed by Tricine-SDS PAGE and Western blot (specific for His-tagged proteins) according to the protocol specified in the <i>General protocols</i> section.</li><br>
+
-
</ol>
+
-
   
+
-
<div style="position:relative"><a class="ancla" name="HYPP"></a></div>
+
-
<h2>Hypoxic Promoters Characterization Protocol</h2>
+
-
 
+
-
<p>This module aims to observe the induction of promoters under anoxic and aerobic conditions, to prove their functionality.</p>
+
-
 
+
-
<h3>Transformation and Confirmation</h3>
+
-
 
+
-
<p>The transformation and confirmation protocol were followed as established in the General protocols section. The strains were transformed with the expression cassettes:</p>
+
-
<ul class="texto">
+
-
<li>pFG: FNR promoter GFP</li>
+
-
<li>pnG: nirB promoter GFP</li>
+
-
<li>pHG: HIP-1 promoter GFP</li>
+
-
</ul>
+
-
<h3>Promoter Characterization (pHG)</h3>
+
-
<ol>
+
-
<li>Six groups of two Corning® 50mL tubes were inoculated with pHG cells, each one in aerobic (tube with a swab) and anoxic (totally closed) conditions.</li>
+
-
<br>
+
-
<li>Each one of the six groups was grown for different periods of time (4h, 8h, 24h, 48h, 72h and 96h) at 37&#176;C and 250 rpm.</li>
+
-
<br>
+
-
<li>After each assigned period of incubation, the cells were harvested and lysed according to the cell rupture protocol (General protocols section).</li>
+
-
<br>
+
-
<li>The protein concentration of the soluble fraction was measured by Biuret method (200µL of sample + 800µL of Biuret reagent incubated for 15 minutes at room temperature).</li>
+
-
<br>
+
-
<li>The samples were plated in a 96-well microplate according to the following layout to determine the fluorescence:
+
-
<img src="https://static.igem.org/mediawiki/igem.org/2/2f/Metodo15.png" style="width:900px;height:180px;">
+
-
    <br>
+
-
</li>
+
-
<li>The microplate was read in the Synergy HT KC4 BioTek fluorescent microplate reader. The emission wavelength was 525nm and the excitation wavelength was 485 nm.</li>
+
-
    <br>
+
-
</ol>
+
-
<h3>Bioreactor protocol (pFG & pnG)</h3>
+
-
 
+
-
<p>This protocol aims to establish the oxygen concentration in which each of the two promoters induce expression.</p>
+
-
<ol>
+
-
<li>BL21 (DE3) <i>Escherichia coli</i> transformed cells with a GFP coding sequence controlled by a hypoxia inducible promoter, were inoculated in 40 ml LB broth with 100ug/ml of ampicillin and grown overnight at 37&#176;C and 250 rpm.</li>
+
-
<br>
+
-
<li>A bioreactor (Sartorius Biostat ® A plus) with 500 ml of LB broth was inoculated with the overnight culture till 0.1 O.D.600nm and closed to prevent air entry. <br>
+
-
    * The bioreactor was set at 250 rpm. The concentration of oxygen in the liquid was monitored until it got a maximum percentage, this number was established as the maximum concentration of oxygen (100%) that could dissolve in the liquid at the agitation rate indicated above.</li>
+
-
<br>
+
-
<li>The culture was grown for six hours.</li>
+
-
<br>
+
-
<li>A sample of 15 ml of culture was taken every hour monitoring the relative percentage of oxygen.</li>
+
-
<br>
+
-
<li>Cells were harvested and lysed according to the cell rupture protocol (General protocols section).</li>
+
-
<br>
+
-
<li>The concentration of protein was calculated with Biuret method (200µL of sample + 800µL of Biuret reagent incubated for 15 minutes at room temperature).</li>
+
-
<br>
+
-
<li>Fluorescence was measured in a 96 well microplate fluorescence lector (Synergy HT KC4 BioTek) according to the following layout. <br>
+
-
    * The emission wavelength was set to 525nm and the excitation wavelength to 485 nm.
+
-
<br>
+
-
<img src="https://static.igem.org/mediawiki/igem.org/7/74/Metodo16.png"><br>
+
-
    * Promotor can be defined as pFG or pnG.</li>
+
-
</ol>
+
-
<h3>References:</h3>
+
-
<ul class="texto">
+
-
<li>Meerloo, J. v., Kaspers, G. J., & Cloos, J. (2011). Cell Sensitivity Assays: The MTT Assay. Methods in Molecular Biology, 731, 237-245.</li>
+
-
<br>
+
-
<li>Trypsinization of adherent cells. (2002). The cell center. Johns Hopkins University, 1, 2.</li>
+
-
<br>
+
-
<li>Vybrant® MTT Cell Proliferation Assay Kit. (2002, March 27). Life Technologies Protocols. Retrieved May 14, 2013, from es-mx.invitrogen.com/site/mx/es/home/References/protocols/cell-culture/mtt-assay-protocol/vybrant-mtt-cell-proliferation-assay-kit.html</li>
+
-
 
+
-
<li>Silhol, M., et al. (2002). Different Mechanisms For Cellular Internalization Of The HIV-1 Tat-derived Cell Penetrating Peptide and Recombinant Proteins Fused to Tat. European Journal of Biochemistry, 269(2), 494-501.</li>
+
-
<br>
+
-
<li>Trypsinization of adherent cells. (2002). The cell center. Johns Hopkins University, 1, 2.</li>
+
-
<br>
+
-
<li>HisPur Ni-NTA Spin Purification Kit. (n.d.). Thermo scientific. Retrieved September 27, 2013, from http://www.piercenet.com/instructions/2162206.pdf</li>
+
-
<br>
+
-
<li>Pierce® Fast Western Blot Kit, ECL Substrate. (n.d.). Thermo scientific. Retrieved September 27, 2013, from http://www.piercenet.com/instructions/2162133.pdf</li>
+
-
<br>
+
-
<li>Schägger, H. (2006). Tricine–SDS-PAGE. Nature protocols, 1(1), 16-22.</li>
+
-
</ul>
+
-
    </div>
+
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 +
            var html_calendario = [
 +
                    "<h3 class =\"mes\">Week 1</h3><div class =\"parrafo\"><h4 class =\"texto\">February 27 2013</h4><ul class =\"texto\"><li>Search for new ideas to work on</li><li>Work on letter for future sponsors</li></ul></div><div class =\"parrafo\"></div>",
 +
                    "<h3 class =\"mes\">Week 2</h3><div class =\"parrafo\"><h4 class =\"texto\">April 1 2013</h4><ul class =\"texto\"><li>Research for type of bacteria</li><ul><li>Salmonella</li><li>Clostridium</li><li>E.Coli</li></ul></ul><h4 class =\"texto\">April 7 2013</h4><p class =\"texto\">Research for DNA sequences</p></div><div class =\"parrafo\"></div>",
 +
                    "<h3 class =\"mes\">Week 3</h3><div class =\"parrafo\"><h4 class =\"texto\">May 26 2013</h4><p class =\"textosub\">Logistics</p><ul class =\"textos\"><li>Send constructs to GenScript</li><li>Quote Materials</li><li>Make a calendar for all the tests</li></ul><p class =\"textosub\">Lab</p><p class =\"texto\">Put strains (BL21, TOP10F, ROSETTA) on -80°C refrigerator</p><ul class =\"textos\"><li>Stock og 5ml AMP (100mg/mC)</li><li>Prepare 200ml LB liquid</li><li>Steralize pipette tips and tubes of 50ml and 30 ml</li></ul><h4 class =\"texto\">May 27 2013</h4><p class =\"textosub\">Logistics</p><ul class =\"textos\"><li>Send sequences to GenScript</li><li>Make a calendar for teams'vacations</li></ul></div><div class =\"parrafo\"><p class =\"textosub\">Lab</p><p class =\"texto\">Competent cells preparation</p><h4 class =\"texto\">May 29 2013</h4><p class =\"textosub\">Lab</p><p class =\"texto\">Competent cells preparation</p><h4 class =\"texto\">May 30 2013</h4><p class =\"textosub\">Lab</p><p class =\"texto\">Competent cells preparation</p></div>",
 +
                    "<h3 class =\"mes\">Week 4</h3><div class =\"parrafo\"><h4 class =\"texto\">June 5 2013</h4><p class =\"textosub\">Logistics</p><p class =\"texto\">Send constructs to GenScript</p><p class =\"textosub\">Lab</p><ul class =\"texto\"><li>Miniprep Puc18</li><li>Electrophoresis</li><li>Cell transformation</li></ul><h4 class =\"texto\">June 6 2013</h4><p class =\"textosub\">Lab</p><ul class =\"texto\"><li>Miniprep Puc18</li><li>Electrophoresis pUc18</li><li>Cels Stock with pUc18</li></ul></div><div class =\"parrafo\"><h4 class =\"texto\">June 7 2013</h4><p class =\"textosub\">Lab</p><ul class =\"texto\"><li>puc18 Transformation</li><li>Electrophoresis</li></ul></div>",
 +
                    "<h3 class =\"mes\">Week 5</h3><div class =\"parrafo\"><h4 class =\"texto\">June 10 2013</h4><p class =\"textosub\">Lab</p><ul class =\"texto\"><li>Digestion</li></ul><h4 class =\"texto\">June 11 2013</h4><p class =\"textosub\">Lab</p><ul class =\"texto\"><li>Sterilize material</li><li>Transformation</li></ul></div><div class =\"parrafo\"><h4 class =\"texto\">June 12 2013</h4><p class =\"textosub\">Lab</p><ul class =\"texto\"><li>Mini Preparation of ADN</li></ul><h4 class =\"texto\">June 13 2013</h4><p class =\"textosub\">Lab</p><ul class =\"texto\"><li>Digestion</li><li>Electrophoresis</li></ul></div>",
 +
                    "<h3 class =\"mes\">Week 6</h3><div class =\"parrafo\"><h4 class =\"texto\">June 17 2013</h4><p class =\"textosub\">Lab</p><p class =\"texto\">Digestion</p><h4 class =\"texto\">June 18 2013</h4><p class =\"textosub\">Lab</p><ul class =\"textos\"><li>Miniprep 3G815l2011</li><li>Nanodrop</li><li>Digestion</li><li>Electrophoresis</li></ul><p class =\"textosub\">Logistics</p><ul class =\"textos\"><li>Work with protocoles</li><li>Search for ways to raise money</li><li>Sponsors</li><li>ideo</li></ul></div><div class =\"parrafo\"><h4 class =\"texto\">June 19 2013</h4><p class =\"textosub\">Lab</p><p class =\"texto\">Mini Preparation of ADN</p><h4 class =\"texto\">June 20 2013</h4><p class =\"textosub\">Lab</p><ul class =\"textos\"><li>Competent cells</li><li>Digestion</li><li>Electrophoresis Gel</li></ul><h4 class =\"texto\">June 21 2013</h4><p class =\"textosub\">Lab</p><ul class =\"textos\"><li>Mini prep</li><li>Digestion</li><li>Electrophoresis Gel</li><li>Transformation</li></ul></div>",
 +
                    "<h3 class =\"mes\">Week 7</h3><div class =\"parrafo\"><h4 class =\"texto\">June 24 2013</h4><p class =\"textosub\">Lab</p><ul class =\"texto\"><li>Digestion 15LA and 15LB</li><li>Electrophoresis</li><li>Selecto colonies of K395602</li><li>Sterilize</li><li>Prepare bromide</li><li>Transformation J04450</li></ul><h4 class =\"texto\">June 26 2013</h4><p class =\"textosub\">Lab</p><ul class =\"texto\"><li>Transfomration with Psb3k3</li><li>Leave inoculum RFP</li><li>Prepare material for competent cells</li><li>Leave inoculim BL21</li></ul></div><div class =\"parrafo\"><h4 class =\"texto\">June 27 2013</h4><p class =\"textosub\">Lab</p><ul class =\"texto\"><li>Inoculate with transformation cells</li><li>Mni prep</li></ul><h4 class =\"texto\">June 28 2013</h4><p class =\"textosub\">Lab</p><ul class =\"texto\"><li>Digestion</li><li>Electrophoresis</li></ul></div>",
 +
                    "<h3 class =\"mes\">Week 8</h3><div class =\"parrafo\"><h4 class =\"texto\">Jly 8 2013</h4><p class =\"textosub\">Lab</p><ul class =\"texto\"><li>Prepare arabinose stocks</li><li>Prepare stick IPGT</li><li>Sterilize LB</li><li>Change cells to LB</li><li>induce cells</li><ul class =\"texto\"><li>IPTG 0.5 mM</li><li>ARA 0.5% w/w</li></ul><li>Incubate cells 37°C 4 hours</li><li>Pelet cells and store @-20°C</li></ul></div><div class =\"parrafo\"><h4 class =\"texto\">July 9 to July 11 2013</h4><p class =\"textosub\">Lab</p><p class =\"texto\">Practice SDS PAGE</p></div>",
 +
                    "<h3 class =\"mes\">Week 9</h3><div class =\"parrafo\"><h4 class =\"texto\">July 15 2013</h4><p class =\"textosub\">Lab</p><p class =\"texto\">Repeat and Practice SDS PAGE</p><h4 class =\"texto\">July 22 2013</h4><p class =\"textosub\">Lab</p><ul class =\"texto\"><li>Learn how to use bioreactor</li><li>Calibrate bioreactor</li></ul></div><div class =\"parrafo\"></div>",
 +
                    "<h3 class =\"mes\">Week 10</h3><div class =\"parrafo\"><h4 class =\"texto\">July 31 2013</h4><p class =\"textosub\">Lab</p><ul class =\"textos\"><li>Received our genes from GenScript</li><li>Prepare material</li><li>Resuspend DNA</li><li>Transformation in top10</li><ul class =\"texto\"><li>phG</li><li>pnG</li><li>pFG</li><li>Tr</li><li>TrH</li><li>A</li><li>S</li><li>GH</li><li>G</li><li>TG</li><li>HG</li><li>Bl21puc</li><li>Top10puc</li><li>l21</li><li>Top10</li></ul></ul></div><div class =\"parrafo\"><h4 class =\"texto\">August 1 2013</h4><p class =\"textosub\">Lab</p><ul class =\"textos\"><li>See the results from transformations</li><li>Transformation in BL21</li></ul><h4 class =\"texto\">August 2 2013</h4><p class =\"textosub\">Lab</p><p class =\"texto\">Mini prep in top10</p><h4 class =\"texto\">August 3 2013</h4><p class =\"textosub\">Lab</p><ul class =\"textos\"><li>Miniprep</li><li>Digestion</li><li>Electrophoresis</li><li>Cells stocks</li><li>Transformation</li></ul></div>",
 +
                    "<h3 class =\"mes\">Week 11</h3><div class =\"parrafo\"><h4 class =\"texto\">August 5 2013</h4><p class =\"textosub\">Lab</p><ul class =\"texto\"><li>Transformations</li><li>Pick colonies</li></ul><h4 class =\"texto\">August 6 2013</h4><p class =\"textosub\">Lab</p><ul class =\"texto\"><li>Miniprep</li><li>Digestion</li><li>Electrophoresis</li><li>Purification of DNA from gel</li><li>SDS page</li><li>Ligation</li></ul></div><div class =\"parrafo\"><h4 class =\"texto\">August 7 2013</h4><p class =\"textosub\">Lab</p><ul class =\"texto\"><li>Gel purification</li><li>Ligation</li><li>SDS page</li></ul></div>",
 +
                    "<h3 class =\"mes\">Week 12</h3><div class =\"parrafo\"><h4 class =\"texto\">August 12 2013</h4><p class =\"textosub\">Lab</p><ul class =\"textos\"><li>Induction Tr, Trh, A</li><li>Inoculation 3 tubes with 15mL LB</li><li>Incubation</li></ul><h4 class =\"texto\">August 13 2013</h4><p class =\"textosub\">Lab</p><p class =\"texto\">Induction of therapeutic proteins</p><h4 class =\"texto\">August 14 2013</h4><p class =\"textosub\">Lab</p><ul class =\"textos\"><li>Pellets</li><li>Digestion</li><li>SDS page</li><li>Electrophoresis</li></ul></div><div class =\"parrafo\"><h4 class =\"texto\">August 16 2013</h4><p class =\"textosub\">Lab</p><ul class =\"texto\"><li>Miniprep</li><li>Digestion</li><li>Electrophoresis</li></ul><h4 class =\"texto\">August 17 2013</h4><p class =\"textosub\">Lab</p><ul class =\"texto\"><li>Purification of proteins with his tag</li><li>SDS page</li><li>Silver stain</li></ul></div>",
 +
                    "<h3 class =\"mes\">Week 13</h3><div class =\"parrafo\"><h4 class =\"texto\">August 21 2013</h4><p class =\"textosub\">Lab</p><ul class =\"texto\"><li>Lysis by sonication</li><li>Miniprep</li><li>Digestion</li><li>Electrophoresis gel</li><li>Lysis and insoluble treatments</li></ul></div><div class =\"parrafo\"><h4 class =\"texto\">August 22 2013</h4><p class =\"textosub\">Lab</p><ul class =\"texto\"><li>Miniprep</li><li>Digestion</li><li>Ligation (A, Tr, PSBIC3)</li><li>Transformation BL21</li></ul></div>",
 +
                    "<h3 class =\"mes\">Week 14</h3><div class =\"parrafo\"><h4 class =\"texto\">August 26 2013</h4><p class =\"textosub\">Lab</p><p class =\"texto\">Electroporation</p><h4 class =\"texto\">August 27 2013</h4><p class =\"textosub\">Lab</p><p class =\"texto\">Digestion</p></div><div class =\"parrafo\"><h4 class =\"texto\">August 28 2013</h4><p class =\"textosub\">Lab</p><ul class =\"texto\"><li>Minipreps</li><li>Confirmation</li><li>Transformation</li><li>Stocks</li><li>Inoculate</li><li>Induce</li></ul></div>",
 +
                    "<h3 class =\"mes\">Week 15</h3><div class =\"parrafo\"><h4 class =\"textos\">September 2 2013</h4><p class =\"textosubs\">Lab</p><p class =\"textos\">Preinoculum (G+S,GH+S,GBHS)</p><h4 class =\"textos\">September 3 2013</h4><p class =\"textosubs\">Lab</p><ul class =\"textos\"><li>Preinoculum</li><li>Inoculate</li></ul><h4 class =\"textos\">September 4</h4><p class =\"textosubs\">Lab</p><ul class =\"textos\"><li>Inoculate apoptine flasks and trail (G+S,GH+S,GBHS)</li><li>Inoculate flasks</li><li>GFP concentrate</li><li>Iduction of TRAIL and apoptin</li><li>Pellets</li><li>Induce</li></ul><h4 class =\"textos\">September 5</h4><p class =\"textosubs\">Lab</p><ul class =\"textos\"><li>Cellular lysis</li><li>Separation of soluble and insoluble fractions</li><li>Protein cuantification</li><li>GFP reading</li></ul></div><div class =\"parrafo\"><h4 class =\"textos\">September 6</h4><p class =\"textosubs\">Lab</p><ul class =\"textos\"><li>Lysis</li><li>Sample concentrations</li></ul><h4 class =\"textos\">September 7</h4><p class =\"textosubs\">Lab</p><ul class =\"textos\"><li>SDS PAGE</li><li>Miniprep</li><li>Digestion</li><li>Agarose gel</li><li>Confirmation of BI21 parts</li><li>Flourescence reading 96 well plates</li></ul><h4 class =\"textos\">September 8</h4><p class =\"textosubs\">Lab</p><ul class =\"textos\"><li>Apoptin and trail SDS PAGE DE</li><li>Hypoxia promoters SDS Page</li><li>Treansformation of BLB21pLysS & c41pGH</li></ul></div>",
 +
                    "<h3 class =\"mes\">Week 16</h3><div class =\"parrafo\"><h4 class =\"texto\">September 12 2013</h4><p class =\"textosub\">Logistics</p><p class =\"texto\">Doughnuts sale for fundraising</p><h4 class =\"texto\">September 13 2013</h4><p class =\"textosub\">Lab</p><ul class =\"texto\"><li>Inoculate for biobricks</li><li>Test in bioreactor</li></ul></div><div class =\"parrafo\"><h4 class =\"texto\">September 14</h4><p class =\"textosub\">Lab</p><ul class =\"texto\"><li>Miniprep</li><li>IDigestion</li><li>Electrophoresis for biobricks</li><li>BIoreacto test</li><li>Proteins induction</li></ul><h4 class =\"texto\">September 15</h4><p class =\"textosub\">Lab</p><ul class =\"texto\"><li>Cell lysis</li><li>Proteins induction</li><li>Flourescence reading 96 well plates</li></ul></div>",
 +
                    "<h3 class =\"mes\">Week 17</h3><div class =\"parrafo\"><h4 class =\"textos\">September 16 2013</h4><p class =\"textosubs\">Lab</p><ul class =\"textos\"><li>Miniprep</li><li>Digestion</li><li>Electrophoresis</li><li>Cell lysis</li><li>Florescence reading 96 well plates</li><li>Protein purification</li></ul><h4 class =\"textos\">September 17</h4><p class =\"textosubs\">Lab</p><ul class =\"textos\"><li>SDS PAGE</li><li>WESTERN BLOT</li><li>Induction</li><li>Proten purification</li></ul><h4 class =\"textos\">September 18</h4><p class =\"textosubs\">Lab</p><ul class =\"textos\"><li>Retransformate TG</li><li>Inoculate TG's</li><li>Inoculate pFG,pHG,pnG and BL21</li></ul></div><div class =\"parrafo\"><h4 class =\"textos\">September 19</h4><p class =\"textosubs\">Lab</p><ul class =\"textos\"><li>Pick 5 colonies</li><li>Inoculate</li></ul><h4 class =\"textos\">September 20</h4><p class =\"textosubs\">Lab</p><ul class =\"textos\"><li>Inoculate with TG</li><li>Incubate at 37°C</li><li>Induction</li><li>Pelletize and sonicate</li><li>Biuret</li><li>Purify samples</li></ul><h4 class =\"textos\">September 21</h4><p class =\"textosubs\">Lab</p><ul class =\"textos\"><li>Buiret</li><li>SDSPAGE</li></ul></div>",
 +
                    "<h3 class =\"mes\">Week 18</h3><div class =\"parrafo\"><h4 class =\"textos\">September 22 2013</h4><p class =\"textosubs\">Lab</p><p class =\"textos\">Concentration and purification of samples</p><h4 class =\"textos\">September 23 2013</h4><p class =\"textosubs\">Lab</p><ul class =\"textos\"><li>BLOTTING</li><li>WESTERN BLOT</li></ul><h4 class =\"textos\">September 24 2013</h4><p class =\"textosubs\">Lab</p><ul class =\"textos\"><li>Prepare inoculi for induction</li><li>Mammal cell essay with therapeutic proteins</li></ul></div><div class =\"parrafo\"><h4 class =\"textos\">September 25</h4><p class =\"textosubs\">Lab</p><ul class =\"textos\"><li>Induction</li><li>Cell harvesting</li><li>Mammal cell essay with internalization proteins</li><li>Protein purification and concentration</li><li>WESTERN BLOT</li><li>Cell harvesting</li>Flourescence reading</ul><h4 class =\"textos\">September 26</h4></ul><p class =\"textosubs\">Lab</p><p class =\"textos\">Doughnut sale for fundraising</p><p class =\"textosubs\">Lab</p><ul class =\"textos\"><li>MTT essay</li><li>Flourescence reading</li></div>"
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Latest revision as of 03:44, 29 October 2013

iGem mty

return

week 1

FEB 27

week 2

APR 1 - APR 7

week 3

MAY 26 - MAY 30

week 4

JUN 5 - JUN 7

week 5

JUN 10 - JUN 13

week 6

JUN 17 - JUN 21

week 7

JUN 24 - JUN 28

week 8

JUL 8 - JUL 11

week 9

JUL 15 - JUL 22

week 10

JUL 31 - AUG 3

week 11

AUG 5 - AUG 7

week 12

AUG 12 - AUG 17

week 13

AUG 21 - AUG 22

week 14

AUG 26 - AUG 28

week 15

SEP 2 - SEP 8

week 16

SEP 12 - SEPT 15

week 17

SEP 16 - SEP 21

week 18

SEP 22 - SEPT 26

Retrieved from "http://2013.igem.org/Team:TecMonterrey/methods.html"