Team:Marburg/Notebook:October
From 2013.igem.org
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<!-- Light-inducible promotor --> | <!-- Light-inducible promotor --> | ||
- | <fieldset class="experiment | + | <fieldset class="experiment analylight"> |
<legend>Light-inducible promoter</legend> | <legend>Light-inducible promoter</legend> | ||
<div class="investigator"> | <div class="investigator"> | ||
Line 20: | Line 20: | ||
<div class="aim"> | <div class="aim"> | ||
<span class="aim">Aim:</span> | <span class="aim">Aim:</span> | ||
- | <span class="aim-desc">Characterization of the light-inducible promotor P<sub>fcpB</sub> (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1071003 | + | <span class="aim-desc">Characterization of the light-inducible promotor P<sub>fcpB</sub> (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1071003">BBa_K1071003</a>).</span> |
</div> | </div> | ||
<div class="exp-content"> | <div class="exp-content"> | ||
Line 43: | Line 43: | ||
<div class="aim"> | <div class="aim"> | ||
<span class="aim">Aim:</span> | <span class="aim">Aim:</span> | ||
- | <span class="aim-desc">Test of disruption method | + | <span class="aim-desc">Test of glass beads disruption method. |
</span> | </span> | ||
</div> | </div> | ||
Line 52: | Line 52: | ||
<li>0.5 ml glass beads were added.</li> | <li>0.5 ml glass beads were added.</li> | ||
<li>Tubes were shaken for 3 min → color shift from brown to green.</li> | <li>Tubes were shaken for 3 min → color shift from brown to green.</li> | ||
- | <li>TCA precipitation was performed.</li> | + | <li>Trichloracetic acid (TCA) precipitation was performed.</li> |
</ul> | </ul> | ||
- | + | <p> | |
- | + | <table class="abtable"> | |
- | + | <colgroup> | |
- | + | <col width="48%" /> | |
+ | <col width="4%" /> | ||
+ | <col width="48%" /> | ||
+ | </colgroup> | ||
+ | <thead> | ||
+ | <th colspan="3">Composition of the phosphate-buffered saline (PBS) buffer:</th> | ||
+ | </thead> | ||
+ | <tr> | ||
+ | <td>Sodium chloride</td> | ||
+ | <th> </th> | ||
+ | <td>137 mM</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Potassium chloride</td> | ||
+ | <th> </th> | ||
+ | <td>2.7 mM</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Na2HPO4</td> | ||
+ | <th> </th> | ||
+ | <td>10 mM</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>KH2PO4</td> | ||
+ | <th> </th> | ||
+ | <td>1.8 mM</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </p> | ||
</div> | </div> | ||
</fieldset> | </fieldset> | ||
+ | |||
+ | <!-- Cell disruption --> | ||
+ | <fieldset class="experiment sonification"> | ||
+ | <legend><a name="soni">Cell disruption by means of ball mill</a></legend> | ||
+ | <div class="investigator"> | ||
+ | <span class="inv">Investigator:</span> | ||
+ | <span class="inv-names">Domenica</span> | ||
+ | </div> | ||
+ | <div class="aim"> | ||
+ | <span class="aim">Aim:</span> | ||
+ | <span class="aim-desc">Test of ball mill disruption method. | ||
+ | </span> | ||
+ | </div> | ||
+ | <div class="exp-content"> | ||
+ | <p><ul class="digest"> | ||
+ | <li>4 ml cell culture was harvested.</li> | ||
+ | <li>Dilution in 300 µL 8M urea buffer.</li> | ||
+ | <li>Shock freezing of suspension by means of liquid nitrogen.</li> | ||
+ | <li>Two steel balls were added → Tube was shock freezed again.</li> | ||
+ | <li>Cell disruption was carried out by 3 times shaking for 30 sec and another shock freezing.</li> | ||
+ | </ul> | ||
+ | |||
+ | <p> | ||
+ | <table class="abtable"> | ||
+ | <colgroup> | ||
+ | <col width="48%" /> | ||
+ | <col width="4%" /> | ||
+ | <col width="48%" /> | ||
+ | </colgroup> | ||
+ | <thead> | ||
+ | <th colspan="3">Composition of the urea buffer (for 40 ml):</th> | ||
+ | </thead> | ||
+ | <tr> | ||
+ | <td>Urea 8 M</td> | ||
+ | <th> </th> | ||
+ | <td>19,22 g</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2 M Tris-HCl pH 6,8</td> | ||
+ | <th> </th> | ||
+ | <td>4 ml</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>0,5 M EDTA</td> | ||
+ | <th> </th> | ||
+ | <td>8 µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10 % SDS</td> | ||
+ | <th> </th> | ||
+ | <td>20 ml</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Bromophenol blue</td> | ||
+ | <th> </th> | ||
+ | <td>12 mg</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </p> | ||
+ | |||
+ | </div> | ||
+ | </fieldset> | ||
+ | |||
+ | <!-- Cell disruption --> | ||
+ | <fieldset class="experiment sonification"> | ||
+ | <legend><a name="soni">Cell disruption by means of sonication</a></legend> | ||
+ | <div class="investigator"> | ||
+ | <span class="inv">Investigator:</span> | ||
+ | <span class="inv-names">Franzi</span> | ||
+ | </div> | ||
+ | <div class="aim"> | ||
+ | <span class="aim">Aim:</span> | ||
+ | <span class="aim-desc">Test of sonication disruption method. | ||
+ | </span> | ||
+ | </div> | ||
+ | <div class="exp-content"> | ||
+ | <p><ul class="digest"> | ||
+ | <li>4 ml cell culture was harvested (5 min centrifugation at full speed).</li> | ||
+ | <li>The supernatent was discarded and the pellet frozen in liquid nitrogen.</li> | ||
+ | <li>The frozen pellet was resuspended in 0.68 ml IP buffer and sonicated (48 cycles, 30 sec on, 30 sec off, 4°C, high).</li> | ||
+ | <li>Extract was centrifuged at 7000 rcf for 5 min, the supernatant was transferred in a new reaction tube.</li> | ||
+ | <li>1,7 µl supernatant of cell lysis were mixed with 350 µl 70 % TCA and incubated for 20 min on ice.</li> | ||
+ | <li>The mixture was centrifuged for 15 min at 20.000 rcf and the pellet washed twice with 1 ml acetone.</li> | ||
+ | <li>The pellet was dried at RT and resuspended in 60 µl 8M ureabuffer (15 min 60 °C).</li> | ||
+ | </ul> | ||
+ | <p> | ||
+ | <table class="abtable"> | ||
+ | <colgroup> | ||
+ | <col width="48%" /> | ||
+ | <col width="4%" /> | ||
+ | <col width="48%" /> | ||
+ | </colgroup> | ||
+ | <thead> | ||
+ | <th colspan="3">Composition of the immuno precipitation (IP) buffer:</th> | ||
+ | </thead> | ||
+ | <tr> | ||
+ | <td>HEPES-KOH buffer pH 7.5</td> | ||
+ | <th> </th> | ||
+ | <td>50 mM</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Sodium chloride</td> | ||
+ | <th> </th> | ||
+ | <td>150 mM</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>EDTA</td> | ||
+ | <th> </th> | ||
+ | <td>1 mM</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Triton X 100</td> | ||
+ | <th> </th> | ||
+ | <td>1 %</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Sodium deoxycholate</td> | ||
+ | <th> </th> | ||
+ | <td>0.1 %</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Sodium dodecyl sulfate (SDS)</td> | ||
+ | <th> </th> | ||
+ | <td>0.1 %</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </p> | ||
+ | </div> | ||
+ | </fieldset> | ||
+ | |||
+ | <!-- Light-inducible promotor --> | ||
+ | <fieldset class="experiment abprod"> | ||
+ | <legend>SDS-PAGE</legend> | ||
+ | <div class="investigator"> | ||
+ | <span class="inv">Investigator:</span> | ||
+ | <span class="inv-names">Dominik</span> | ||
+ | </div> | ||
+ | <div class="aim"> | ||
+ | <span class="aim">Aim:</span> | ||
+ | <span class="aim-desc">Identification of the most efficient disruption technique.</span> | ||
+ | </div> | ||
+ | <div class="exp-content"> | ||
+ | <p>20 µl of each cell suspension obtained from the three different disruption techniques were load on a 12 % SDS gel.</p> | ||
+ | <p> | ||
+ | <table class="abtable"> | ||
+ | <colgroup> | ||
+ | <col width="48%" /> | ||
+ | <col width="4%" /> | ||
+ | <col width="48%" /> | ||
+ | </colgroup> | ||
+ | <thead> | ||
+ | <th colspan="3">Separation gel</th> | ||
+ | </thead> | ||
+ | <tr> | ||
+ | <td>1 M Tris-HCl pH8,8</td> | ||
+ | <th> </th> | ||
+ | <td>2,25 ml</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Aa/Bis 30:0,88</td> | ||
+ | <th> </th> | ||
+ | <td>2,4 ml</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>H<sub>2</sub>O</td> | ||
+ | <th> </th> | ||
+ | <td>1,2 ml</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10 % SDS (w/v)</td> | ||
+ | <th> </th> | ||
+ | <td>75 µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10 % APS</td> | ||
+ | <th> </th> | ||
+ | <td>100 µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>TEMED</td> | ||
+ | <th> </th> | ||
+ | <td>10 µl</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </p> | ||
+ | <p> | ||
+ | <table class="abtable"> | ||
+ | <colgroup> | ||
+ | <col width="48%" /> | ||
+ | <col width="4%" /> | ||
+ | <col width="48%" /> | ||
+ | </colgroup> | ||
+ | <thead> | ||
+ | <th colspan="3">Stacking gel</th> | ||
+ | </thead> | ||
+ | <tr> | ||
+ | <td>1 M Tris-HCl pH6,8</td> | ||
+ | <th> </th> | ||
+ | <td>375 µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Aa/Bis 30:0,88</td> | ||
+ | <th> </th> | ||
+ | <td>500 µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>H<sub>2</sub>O</td> | ||
+ | <th> </th> | ||
+ | <td>2050 µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10 % SDS (w/v)</td> | ||
+ | <th> </th> | ||
+ | <td>30 µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10 % APS</td> | ||
+ | <th> </th> | ||
+ | <td>50 µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>TEMED</td> | ||
+ | <th> </th> | ||
+ | <td>7,5 µl</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </p> | ||
+ | |||
+ | <!-- Gel-Bild--> | ||
+ | <table class="gel pcr"> | ||
+ | <colgroup> | ||
+ | <col width="50%" /> | ||
+ | <col width="50%" /> | ||
+ | </colgroup> | ||
+ | <thead> | ||
+ | <tr> | ||
+ | <th colspan="2" class="title">SDS gel</th> | ||
+ | </tr> | ||
+ | </thead> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/c/c8/Mr_disruption_methods_2110.png" width="100%" alt="gel-electrophoresis-image" /> | ||
+ | </td> | ||
+ | <td> | ||
+ | <p> | ||
+ | <span class="gel-elc">Gel substances</span> | ||
+ | <ul class="gel-sub"> | ||
+ | <li>12 % Acryl amide gel</li> | ||
+ | <li>6 µl ColorPlus Prestained Protein Ladder (NEB)</li> | ||
+ | </p> | ||
+ | |||
+ | |||
+ | <table class="gel"> | ||
+ | <colgroup> | ||
+ | <col width="9%" /> | ||
+ | <col width="2%" /> | ||
+ | <col width="42%" /> | ||
+ | <col width="2%" /> | ||
+ | <col width="45%" /> | ||
+ | </colgroup> | ||
+ | <thead> | ||
+ | <th>Lane</th> | ||
+ | <th> </th> | ||
+ | <th>Content</th> | ||
+ | <th> </th> | ||
+ | <th>Expectations</th> | ||
+ | </thead> | ||
+ | <tr> | ||
+ | <td>2</td> | ||
+ | <th> </th> | ||
+ | <td>Ball mill + protease inh.</td> | ||
+ | <th> </th> | ||
+ | <td>26 kDa (lc) + 55 kDa (hc)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3</td> | ||
+ | <th> </th> | ||
+ | <td>Ball mill</td> | ||
+ | <th> </th> | ||
+ | <td>26 kDa (lc) + 55 kDa (hc)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4</td> | ||
+ | <th> </th> | ||
+ | <td>Glass beads + prot. inh.</td> | ||
+ | <th> </th> | ||
+ | <td>26 kDa (lc) + 55 kDa (hc)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5</td> | ||
+ | <th> </th> | ||
+ | <td>Glass beads</td> | ||
+ | <th> </th> | ||
+ | <td>26 kDa (lc) + 55 kDa (hc)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>6</td> | ||
+ | <th> </th> | ||
+ | <td>Sonication (total)</td> | ||
+ | <th> </th> | ||
+ | <td>26 kDa (lc) + 55 kDa (hc)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>7</td> | ||
+ | <th> </th> | ||
+ | <td>Sonication (SN)</td> | ||
+ | <th> </th> | ||
+ | <td>26 kDa (lc) + 55 kDa (hc)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>8</td> | ||
+ | <th> </th> | ||
+ | <td>Sonication (P)</td> | ||
+ | <th> </th> | ||
+ | <td>26 kDa (lc) + 55 kDa (hc)</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | |||
+ | <p>Result: The highest amounts of proteins were gained using sonication and the ball mill.</p> | ||
+ | </div> | ||
+ | </fieldset> | ||
+ | |||
+ | <!-- Light-inducible promotor --> | ||
+ | <fieldset class="experiment analylight"> | ||
+ | <legend>Test of antibody production</legend> | ||
+ | <div class="investigator"> | ||
+ | <span class="inv">Investigator:</span> | ||
+ | <span class="inv-names">Dominik</span> | ||
+ | </div> | ||
+ | <div class="aim"> | ||
+ | <span class="aim">Aim:</span> | ||
+ | <span class="aim-desc">Preparation for Western Blotting.</span> | ||
+ | </div> | ||
+ | <div class="exp-content"> | ||
+ | <p>To demonstrate the production of antibodies in the induced cultures 2.1 and 4.2 a Western Blot should be performed. For this purpose, the cells were harvested and the pellet was separated from the supernatant. After that, a TCA precipitation was performed. All samples were stored at -20°C.</p> | ||
+ | </div> | ||
+ | </fieldset> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | <div class="notebooky-entry"> | ||
+ | <h2 class="title"> | ||
+ | <a name="22-10-2013">22.10.2013</a> | ||
+ | </h2> | ||
+ | |||
+ | <!-- Cell disruption --> | ||
+ | <fieldset class="experiment sonification"> | ||
+ | <legend><a name="soni">Cell disruption by means of sonication</a></legend> | ||
+ | <div class="investigator"> | ||
+ | <span class="inv">Investigator:</span> | ||
+ | <span class="inv-names">Franzi</span> | ||
+ | </div> | ||
+ | <div class="aim"> | ||
+ | <span class="aim">Aim:</span> | ||
+ | <span class="aim-desc">Find the best buffer for sonication disruption method. | ||
+ | </span> | ||
+ | </div> | ||
+ | <div class="exp-content"> | ||
+ | <p>To examine whether the PBS buffer or the IP buffer is best for disruption by means of sonication, we performed a sonication with both buffers. Afterwards a TCA precipitation was made and a SDS-PAGE was carried out.</p> | ||
+ | <p>Result: We gained higher amounts of protein by using the IP buffer. Consequently, all following cell disruptions by sonication were performed with IP buffer.</p> | ||
+ | </div> | ||
+ | </fieldset> | ||
+ | |||
+ | <!-- Light-inducible promotor --> | ||
+ | <fieldset class="experiment analylight"> | ||
+ | <legend>Light-inducible promoter</legend> | ||
+ | <div class="investigator"> | ||
+ | <span class="inv">Investigators:</span> | ||
+ | <span class="inv-names">Domenica and Dominik</span> | ||
+ | </div> | ||
+ | <div class="aim"> | ||
+ | <span class="aim">Aim:</span> | ||
+ | <span class="aim-desc">Characterization of the light-inducible promotor P<sub>fcpB</sub> (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1071003">BBa_K1071003</a>).</span> | ||
+ | </div> | ||
+ | <div class="exp-content"> | ||
+ | <table class="gel pcr"> | ||
+ | <colgroup> | ||
+ | <col width="48%" /> | ||
+ | <col width="4%" /> | ||
+ | <col width="48%" /> | ||
+ | </colgroup> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <p>After two days of incubation in darkness, the culture was divided in 5 samples, which were incubated under 5 different light conditions: normal light, green light, red light, blue light (50 µE each) and darkness. The division was realised in darkness to prevent induction of the light-inducible promoter. OD (600 nm): 0.450.</p></td> | ||
+ | <th> </th> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2013/4/41/Mr_lighting_chamber.png" width="100%" alt="lightning chamber" /> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | </div> | ||
+ | </fieldset> | ||
+ | |||
+ | <!-- Light-inducible promotor --> | ||
+ | <fieldset class="experiment analylight"> | ||
+ | <legend>Test of antibody production</legend> | ||
+ | <div class="investigator"> | ||
+ | <span class="inv">Investigator:</span> | ||
+ | <span class="inv-names">Dominik</span> | ||
+ | </div> | ||
+ | <div class="aim"> | ||
+ | <span class="aim">Aim:</span> | ||
+ | <span class="aim-desc">Western Blot of samples before and after induction.</span> | ||
+ | </div> | ||
+ | <div class="exp-content"> | ||
+ | <p>The samples that were taken the previous day and the days before induction with nitrate were used for a 12 % SDS gel (see above) and subsequent Western blotting on a nitro cellulose membrane. To accomplish this four Whatman filter papers were soaked into membrane buffer and a nitro cellulose membrane, also soaked in membrane buffer, were stacked. On this the SDS gel was placed and covered with with four Whatman filter papers soaked with gel buffer. Then the proteins were transferred with 0.8 mA/cm<sup>2</sup> for two hours.</p> | ||
+ | <p>To prevent unspecific binding of the first antibody, the membrane was rotated for at least one hour in 5 % milk powder in TBST at 4 °C. The primary antibody was dissolved in 2 % milk powder in TBST (dillution: 1:500 anti GFP, anti IgG 1:10,000, all antibodies obtained by Santa Cruz biotechnology) and added to the membrane. Afterwards it was washed two times in TBST for five minutes. The secondary antibody was dissolved in 2 % milk powder in TBST (anti anti GFP 1:10,000, anti anti IgG 1:10,000), added to the membrane and incubated for one hour. Afterwards it was washed with TBST for three times for five minutes each. After incubation with a detection agent, containing Luminol, p-Coumaric acid and H2O2, the detection was performed with a Chemocam Professional from INTAS science imaging. </p> | ||
+ | <p> | ||
+ | <table class="abtable"> | ||
+ | <colgroup> | ||
+ | <col width="48%" /> | ||
+ | <col width="4%" /> | ||
+ | <col width="48%" /> | ||
+ | </colgroup> | ||
+ | <thead> | ||
+ | <th colspan="3">Composition of the Tris-buffered saline-tween (TBST) buffer:</th> | ||
+ | </thead> | ||
+ | <tr> | ||
+ | <td>Tris-HCl pH 7.6</td> | ||
+ | <th> </th> | ||
+ | <td>50 mM</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Sodium chloride</td> | ||
+ | <th> </th> | ||
+ | <td>150 mM</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Tween 20</td> | ||
+ | <th> </th> | ||
+ | <td>0.05 %</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </p> | ||
+ | <ul>Sample preparation: | ||
+ | <li>Resuspend pellet in 1 ml water</li> | ||
+ | <li>Measure OD (600 nm)</li> | ||
+ | <li>Take the volume corresponding to a certain OD</li> | ||
+ | <li>Protein preparation of the taken sample with the predefined OD | ||
+ | <ul> | ||
+ | <li>Cell culture is centrifuged for 5 minutes (full speed)</li> | ||
+ | <li>Discard supernatant</li> | ||
+ | <li>Add 150 µl sodium hydroxide</li> | ||
+ | <li>Incubate 10 minutes on ice</li> | ||
+ | <li>Add 850 µl bidest. water</li> | ||
+ | <li>Add 200 µl 70 % TCA and incubate for 20 min on ice</li> | ||
+ | <li>Centrifuge for 15 minutes at 4 °C (20000xg)</li> | ||
+ | <li>Wash pellet with aceton and centrifuge for 15 minutes at 4 °C</li> | ||
+ | <li>Repeat the latter step until the chlorophyll is washed out</li> | ||
+ | <li>Dry pellet at room temperature</li> | ||
+ | <li>Resuspend in 8 M urea buffer</li> | ||
+ | <li>Shake the samples for 15 min at 60 °C</li> | ||
+ | <li>Load samples on a 12 % SDS gel</li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | <li>Load samples on the gel</li> | ||
+ | <li>Run gel for at least 1 h (120V) | ||
+ | <li>Mini-Protean Tetra Cell (Biorad)</li> | ||
+ | <li>Equilibrate the gel in Towbin transfer buffer (25 mM Tris, 192 mM glycine pH 8.3, 20 % MeOH) for 10 min</li> | ||
+ | <li>Blotting on a nitrocellulose membrane using the <b>STANDARD SD</b> transfer protocol from Biorad preprogrammed protocols (Trans-Blot Turbo Blotting System)</li> | ||
+ | <li>Submerge blot in 4 % milk powder in TBST</li> | ||
+ | <li>Incubate for 2 h at 4 °C</li> | ||
+ | <li>Incubate membrane for 1-2 h with the milk and the primary antibody against GFP and tubulin (dilution: 1:1000 GFP, 1:2000 tubulin)</li> | ||
+ | <li>Wash membrane in TBST for 10 min (3 times)</li> | ||
+ | <li>Incubate for 1 h at 4 °C with the secondary antibody in milk-TBST (goat anti mouse HRP)</li> | ||
+ | <li>Wash for 10 min at 4 °C in TBST (3 times)</li> | ||
+ | <li>Detection in western imager station (chemocam) | ||
+ | <ul> | ||
+ | <li>ECL solution: incubate membrane for 1 min in ECL solution</li> | ||
+ | </ul> | ||
+ | </li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | |||
+ | <!-- Gel-Bild--> | ||
+ | <table class="gel pcr"> | ||
+ | <colgroup> | ||
+ | <col width="50%" /> | ||
+ | <col width="50%" /> | ||
+ | </colgroup> | ||
+ | <thead> | ||
+ | <tr> | ||
+ | <th colspan="2" class="title">Western blot 1</th> | ||
+ | </tr> | ||
+ | </thead> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/e/eb/Mr-western_221013.png" width="100%" alt="gel-electrophoresis-image" /> | ||
+ | </td> | ||
+ | <td>In these samples that were taken before the induction, bands at 80 kDa could be detected. They could be due to unspecific bounding of the antibodies.<br> | ||
+ | The second blot, containing the samples that were taken after induction, failed because of a lack of enough transfer buffer.<br> | ||
+ | Additionally, due to insufficient blocking steps, much background signal was observed. For that reason, no statements about the production of antibodies can be made.</td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | |||
+ | </fieldset> | ||
+ | |||
</div> | </div> | ||
Line 67: | Line 600: | ||
</h2> | </h2> | ||
- | <!-- | + | <!-- Light-inducible promotor --> |
+ | <fieldset class="experiment analylight"> | ||
+ | <legend>Test of antibody production</legend> | ||
+ | <div class="investigator"> | ||
+ | <span class="inv">Investigators:</span> | ||
+ | <span class="inv-names">Dominik, Franzi and Domenica</span> | ||
+ | </div> | ||
+ | <div class="aim"> | ||
+ | <span class="aim">Aim:</span> | ||
+ | <span class="aim-desc">Harvesting, cell disruption and TCA prepipitation.</span> | ||
+ | </div> | ||
+ | <div class="exp-content"> | ||
+ | <p>10 mL of the culture were harvested for cell disruption with sonication (see protocol below), 1 mL for cell disruption with the ball mill, and 1 mL as a reserve. | ||
+ | Cells were disrupted with sonication and the ball mill. Optical density (at 600 nm) was measured for each sample. | ||
+ | |||
+ | <table class="gel pcr"> | ||
+ | <colgroup> | ||
+ | <col width="22%" /> | ||
+ | <col width="3%" /> | ||
+ | <col width="22%" /> | ||
+ | <col width="53%" /> | ||
+ | </colgroup> | ||
+ | <thead> | ||
+ | <th>Condition</th> | ||
+ | <th> </th> | ||
+ | <th>OD<sub>600</sub></th> | ||
+ | <th> </th> | ||
+ | </thead> | ||
+ | <tr> | ||
+ | <td>Light</td> | ||
+ | <th> </th> | ||
+ | <td>0.525</td> | ||
+ | <th> </th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Darkness</td> | ||
+ | <th> </th> | ||
+ | <td>0.410</td> | ||
+ | <th> </th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Blue light</td> | ||
+ | <th> </th> | ||
+ | <td>0.576</td> | ||
+ | <th> </th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Red light</td> | ||
+ | <th> </th> | ||
+ | <td>0.505</td> | ||
+ | <th> </th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Green light</td> | ||
+ | <th> </th> | ||
+ | <td>0.552</td> | ||
+ | <th> </th> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </p> | ||
+ | </div> | ||
+ | </fieldset> | ||
+ | |||
+ | <!-- Sonication --> | ||
<fieldset class="experiment sonification"> | <fieldset class="experiment sonification"> | ||
- | <legend><a name="soni"> | + | <legend><a name="soni">Sonication</a></legend> |
<div class="investigator"> | <div class="investigator"> | ||
<span class="inv">Investigators:</span> | <span class="inv">Investigators:</span> | ||
Line 76: | Line 672: | ||
<div class="aim"> | <div class="aim"> | ||
<span class="aim">Aim:</span> | <span class="aim">Aim:</span> | ||
- | <span class="aim-desc">Quantification of the light-inducible promoter P<sub>fcpB</sub> (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1071003 | + | <span class="aim-desc">Quantification of the light-inducible promoter P<sub>fcpB</sub> (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1071003">BBa_K1071003</a>)<br> → Cell disruption, protein precipitation and SDS-PAGE. |
</span> | </span> | ||
</div> | </div> | ||
<div class="exp-content"> | <div class="exp-content"> | ||
<p>10 ml of cultures grown under different light conditions (green, blue, red, full light spectrum and darkness) were harvested in 15 ml Falcon-tubes (5 min centrifugation at full speed). Supernatants were discarded and pellets frozen in liquid nitrogen.</p> | <p>10 ml of cultures grown under different light conditions (green, blue, red, full light spectrum and darkness) were harvested in 15 ml Falcon-tubes (5 min centrifugation at full speed). Supernatants were discarded and pellets frozen in liquid nitrogen.</p> | ||
- | <p>Frozen cell-pellets were resuspended in 1,7 ml IP | + | <p>Frozen cell-pellets were resuspended in 1,7 ml IP buffer and sonicated (48 cycles, 30 sec on, 30 sec off, 4°C, high). Extract was centrifuged at 7000 rcf for 5 min, the supernatant was transferred in a new reaction tube.</p> |
<p>1,7 µl supernatant of cell lysis were mixed with 350 µl 70 % TCA and incubated for 20 min on ice. The mixture was centrifuged for 15 min at 20.000 rcf and the pellet washed twice with 1 ml acetone. The pellet was dried at RT and resuspended in 60 µl 8M ureabuffer (15 min 60 °C).</p> | <p>1,7 µl supernatant of cell lysis were mixed with 350 µl 70 % TCA and incubated for 20 min on ice. The mixture was centrifuged for 15 min at 20.000 rcf and the pellet washed twice with 1 ml acetone. The pellet was dried at RT and resuspended in 60 µl 8M ureabuffer (15 min 60 °C).</p> | ||
<p>10 µg of each sample were used for SDS-PAGE (Westernblot).</p> | <p>10 µg of each sample were used for SDS-PAGE (Westernblot).</p> | ||
Line 87: | Line 683: | ||
</fieldset> | </fieldset> | ||
- | <!-- | + | <!-- Sonication --> |
<fieldset class="experiment amidoblack"> | <fieldset class="experiment amidoblack"> | ||
<legend><a name="amido">Amido black assay</a></legend> | <legend><a name="amido">Amido black assay</a></legend> | ||
<div class="investigator"> | <div class="investigator"> | ||
- | <span class="inv"> | + | <span class="inv">Investigators:</span> |
<span class="inv-names">Franzi, Dominik, Domenica</span> | <span class="inv-names">Franzi, Dominik, Domenica</span> | ||
</div> | </div> | ||
Line 99: | Line 695: | ||
</div> | </div> | ||
<div class="exp-content"> | <div class="exp-content"> | ||
- | <p>5 µl of protein (in | + | <p>5 µl of protein (in urea buffer) from sonication and ball mill disruption were used.</p> |
- | <ul> Measured protein concentrations: | + | <ul>The following protocol was used: |
- | <li>Dark: 2 | + | <li>195 µl H2O</li> |
- | <li>Red: 2 | + | <li>5 µl sample (or 5 µl H2O as negative control)</li> |
- | <li>Green: 2 | + | <li>800 µl amido black solution (0.1 % amido black solution, 90 % MeOH, 10 % acetic acid (100 %))</li> |
- | <li>Blue: 3 | + | <li>Invert 3 times → precipitation of protein-amido black complex</li> |
+ | <li>Centrife: 10 min @ 14,000 rpm</li> | ||
+ | <li>Discard supernatant</li> | ||
+ | <li>Add 1 ml washing solution (90 % EtOH, 10 % acetic acid (100 %))</li> | ||
+ | <li>Centrife: 10 min @ 14,000 rpm</li> | ||
+ | <li>Discard supernatant</li> | ||
+ | <li>Add 1 ml 0.2 M NaOH → complete bleaching of the pellet</li> | ||
+ | </ul> | ||
+ | <p>The protein concentration can be determined photometrically by measuring the absorbance at 615 nm and by comparing the values with a standard curve (created with different amounts of BSA).</p> | ||
+ | <ul> Measured protein concentrations for sonication samples: | ||
+ | <li>Dark: 2.10 µg/µl</li> | ||
+ | <li>Red: 2.49 µg/µl</li> | ||
+ | <li>Green: 2.80 µg/µl</li> | ||
+ | <li>Blue: 3.23 µg/µl</li> | ||
+ | </ul> | ||
+ | <p></p> | ||
+ | <ul> Measured protein concentrations for ball mill samples: | ||
+ | <li>Light: 0.517 µg/µl</li> | ||
+ | <li>Dark: 0.314 µg/µl</li> | ||
+ | <li>Red: 0.639 µg/µl</li> | ||
+ | <li>Green: 0.385 µg/µl</li> | ||
+ | <li>Blue: 0.619 µg/µl</li> | ||
</ul> | </ul> | ||
</div> | </div> | ||
</fieldset> | </fieldset> | ||
+ | <!-- Protein extraction --> | ||
+ | <fieldset class="experiment analylight"> | ||
+ | <legend>Protein extraction from herbal leafs</legend> | ||
+ | <div class="investigator"> | ||
+ | <span class="inv">Investigators:</span> | ||
+ | <span class="inv-names">Domenica, Franzi and Dominik</span> | ||
+ | </div> | ||
+ | <div class="aim"> | ||
+ | <span class="aim">Aim:</span> | ||
+ | <span class="aim-desc">Protein extraktion (positive control).</span> | ||
+ | </div> | ||
+ | <div class="exp-content"> | ||
+ | <p>As a positive control, we extracted a protein-coding gene, <i>cry-gfp</i>, out of <i>Arabidopsis landsberg erecta</i>, which produces GFP as well as tubulin. For this purpose, 228 mg leafs were mixed with 114 µL PEG extraction buffer. Following this, the cells were disrupted with a Polytron PT 1200E Blade-type homogenizer (Brinkmann) and 5 min in a sonication bath. After centrifugation (15 min, 20000g, 4°C), the supernatant was used as protein extract. The concentration of protein was determined using a Bradford assay (Roti Quant, Roth).</p> | ||
+ | <p> | ||
+ | <table class="abtable"> | ||
+ | <colgroup> | ||
+ | <col width="48%" /> | ||
+ | <col width="4%" /> | ||
+ | <col width="48%" /> | ||
+ | </colgroup> | ||
+ | <thead> | ||
+ | <th colspan="3">Composition of the polyethylene glycol (PEG) extraction buffer:</th> | ||
+ | </thead> | ||
+ | <tr> | ||
+ | <td>Tris-HCl pH 7.8</td> | ||
+ | <th> </th> | ||
+ | <td>0.5 M</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Triton X 100</td> | ||
+ | <th> </th> | ||
+ | <td>2 %</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Magnesium chloride</td> | ||
+ | <th> </th> | ||
+ | <td>20 mM</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Phenylmethylsulfonyl fluoride (PMSF)</td> | ||
+ | <th> </th> | ||
+ | <td>1 mM</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Ethylenediaminetetraacetic acid (EDTA) </td> | ||
+ | <th> </th> | ||
+ | <td>1 mM</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Polyvinylpolypyrrolidone (PVPP)</td> | ||
+ | <th> </th> | ||
+ | <td>1 %</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </p> | ||
+ | <p>Result: 5.16 µg/µl protein were extracted.</p> | ||
+ | </div> | ||
+ | </fieldset> | ||
+ | |||
+ | <fieldset class="experiment analylight"> | ||
+ | <legend>Light-inducible promoter: SDS-PAGE and Western blot</legend> | ||
+ | <div class="investigator"> | ||
+ | <span class="inv">Investigator:</span> | ||
+ | <span class="inv-names">Domenica, Franzi and Dominik</span> | ||
+ | </div> | ||
+ | <div class="aim"> | ||
+ | <span class="aim">Aim:</span> | ||
+ | <span class="aim-desc">Quantify the amount of eGFP produced under different light conditions.</span> | ||
+ | </div> | ||
+ | <div class="exp-content"> | ||
+ | <p>5 µg (ball mill) and 10 µg (sonication) protein was used for SDS-PAGE. After that, a Western Blot was performed overnight (4°C, 30V).</p> | ||
+ | </div> | ||
+ | </fieldset> | ||
</div> | </div> | ||
+ | <div class="notebooky-entry"> | ||
+ | <h2 class="title"> | ||
+ | <a name="24-10-2013">24.10.2013</a> | ||
+ | </h2> | ||
+ | |||
+ | <fieldset class="experiment analylight"> | ||
+ | <legend>Light-inducible promoter: Western blot</legend> | ||
+ | <div class="investigator"> | ||
+ | <span class="inv">Investigator:</span> | ||
+ | <span class="inv-names">Domenica, Franzi and Dominik</span> | ||
+ | </div> | ||
+ | <div class="aim"> | ||
+ | <span class="aim">Aim:</span> | ||
+ | <span class="aim-desc">Quantify the amount of eGFP produced under different light conditions.</span> | ||
+ | </div> | ||
+ | <div class="exp-content"> | ||
+ | <p></p> | ||
+ | <ul>After blotting procedure, the following protocol was applied: | ||
+ | <li>Blocking: Incubate in 7 % milk powder in TBST buffer for 1 h at RT</li> | ||
+ | <li>Wash three times for 10 min with TBST buffer at RT</li> | ||
+ | <li>Add the first antibody: goat anti-GFP (1:2000) in TBS with 0.02% NaN3 for 1.5 h at RT</li> | ||
+ | <li>Add the second antiboy: anti-goat 800 (1:10000) in TBS buffer for 1 h at RT</li> | ||
+ | <li>Wash two times for 10 min with TBST buffer at RT</li> | ||
+ | <li>Wash a last time for 10 min with TBS buffer at RT </li> | ||
+ | <li>Detect with scanner at 800 nm</li> | ||
+ | <li>Add the first antibody: mouse anti-tubulin (1:2000) in TBS with 0.02% NaN3 for 1.5 h at RT</li> | ||
+ | <li>Add the second antiboy: anti-mouse 800 (1:10000) in TBS buffer for 1 h at RT</li> | ||
+ | <li>Wash two times for 10 min with TBST buffer at RT</li> | ||
+ | <li>Wash a last time for 10 min with TBS buffer at RT </li> | ||
+ | <li>Detect with scanner at 800 nm</li> | ||
+ | </ul> | ||
+ | <p></p> | ||
+ | |||
+ | <table class="gel pcr"> | ||
+ | <colgroup> | ||
+ | <col width="50%" /> | ||
+ | <col width="50%" /> | ||
+ | </colgroup> | ||
+ | <thead> | ||
+ | <tr> | ||
+ | <th colspan="2" class="title">Western blot</th> | ||
+ | </tr> | ||
+ | </thead> | ||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/5/58/Mr-western-2410.png" width="100%" alt="wester-blot-image" /> | ||
+ | </td> | ||
+ | <td> | ||
+ | <p> | ||
+ | <span class="gel-elc">Gel substances</span> | ||
+ | <ul class="gel-sub"> | ||
+ | <li>12 % Acryl amide gel</li> | ||
+ | <li>6 µl PageRuler Prestained Protein Ladder (Thermo Scientific)</li> | ||
+ | </p> | ||
+ | <p><b>Upper gel</b></p> | ||
+ | |||
+ | |||
+ | <table class="gel"> | ||
+ | <colgroup> | ||
+ | <col width="9%" /> | ||
+ | <col width="3%" /> | ||
+ | <col width="24%" /> | ||
+ | <col width="2%" /> | ||
+ | <col width="62%" /> | ||
+ | </colgroup> | ||
+ | |||
+ | <thead> | ||
+ | <th>Lane</th> | ||
+ | <th> </th> | ||
+ | <th>Content</th> | ||
+ | <th> </th> | ||
+ | <th>Expectations</th> | ||
+ | </thead> | ||
+ | <tr> | ||
+ | <td>2</td> | ||
+ | <th> </th> | ||
+ | <td>Darkness</td> | ||
+ | <th> </th> | ||
+ | <td>27 kDa (GFP) + 55 kDa (tubulin)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3</td> | ||
+ | <th> </th> | ||
+ | <td>Blue light</td> | ||
+ | <th> </th> | ||
+ | <td>27 kDa (GFP) + 55 kDa (tubulin)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4</td> | ||
+ | <th> </th> | ||
+ | <td>Green light</td> | ||
+ | <th> </th> | ||
+ | <td>27 kDa (GFP) + 55 kDa (tubulin)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5</td> | ||
+ | <th> </th> | ||
+ | <td>Red light</td> | ||
+ | <th> </th> | ||
+ | <td>27 kDa (GFP) + 55 kDa (tubulin)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>6</td> | ||
+ | <th> </th> | ||
+ | <td>Control</td> | ||
+ | <th> </th> | ||
+ | <td>55 kDa (tubulin) + 80 kDa ("GFP")</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p><b>Lower gel</b></p> | ||
+ | <table> | ||
+ | <colgroup> | ||
+ | <col width="9%" /> | ||
+ | <col width="3%" /> | ||
+ | <col width="24%" /> | ||
+ | <col width="2%" /> | ||
+ | <col width="62%" /> | ||
+ | </colgroup> | ||
+ | |||
+ | <thead> | ||
+ | <th>Lane</th> | ||
+ | <th> </th> | ||
+ | <th>Content</th> | ||
+ | <th> </th> | ||
+ | <th>Expectations</th> | ||
+ | </thead> | ||
+ | <tr> | ||
+ | <td>8</td> | ||
+ | <th> </th> | ||
+ | <td>Light</td> | ||
+ | <th> </th> | ||
+ | <td>27 kDa (GFP) + 55 kDa (tubulin)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>9</td> | ||
+ | <th> </th> | ||
+ | <td>Darkness</td> | ||
+ | <th> </th> | ||
+ | <td>27 kDa (GFP) + 55 kDa (tubulin)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10</td> | ||
+ | <th> </th> | ||
+ | <td>Blue light</td> | ||
+ | <th> </th> | ||
+ | <td>27 kDa (GFP) + 55 kDa (tubulin)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>11</td> | ||
+ | <th> </th> | ||
+ | <td>Green light</td> | ||
+ | <th> </th> | ||
+ | <td>27 kDa (GFP) + 55 kDa (tubulin)</td> | ||
+ | </tr><tr> | ||
+ | <td>12</td> | ||
+ | <th> </th> | ||
+ | <td>Red light</td> | ||
+ | <th> </th> | ||
+ | <td>27 kDa (GFP) + 55 kDa (tubulin)</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>13</td> | ||
+ | <th> </th> | ||
+ | <td>Control</td> | ||
+ | <th> </th> | ||
+ | <td>55 kDa (tubulin) + 80 kDa ("GFP")</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | |||
+ | <p>Result: All samples showed the expexted bands. For a detailed discussion have a look at our <a href="https://2013.igem.org/Team:Marburg/Project:lightcontrol">light control</a> section.</p> | ||
+ | |||
+ | </div> | ||
+ | </fieldset> | ||
+ | |||
+ | </div> | ||
</html> | </html> | ||
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Latest revision as of 22:50, 28 October 2013