Team:Tsinghua/Notebook-Protocol

From 2013.igem.org

(Difference between revisions)
(Created page with "Notebook-Protocol")
Line 1: Line 1:
-
Notebook-Protocol
+
{{Tsinghua:Common-Style}}
 +
{{Tsinghua:Navigation-Style}}
 +
{{Tsinghua:Navigation-Script}}
 +
<html><body>
 +
<script type="text/javascript">
 +
  window.onload = start;
 +
  </script>
 +
<div id="main">
 +
<div id="header">
 +
<div id="team-logo">
 +
<img height="100%" src="https://static.igem.org/mediawiki/2013/b/b7/Tsinghua-logo.png"/>
 +
</div>
 +
<div id="title-box">
 +
<div id="title-bar">
 +
<img height="100%" src="https://static.igem.org/mediawiki/2013/1/1d/Tsinghua-left-diamond.png"/>
 +
<span id="page-title">Main Page</span>
 +
<img height="100%" src="https://static.igem.org/mediawiki/2013/3/37/Tsinghua-right-diamond.png"/>
 +
</div>
 +
</div>
 +
</div>
 +
<div id="lefter">
 +
<div id="left-three-box">
 +
<img src="https://static.igem.org/mediawiki/2013/9/92/Tsinghua-left-three-box.png" width="100%"/>
 +
</div>
 +
<div id="menu">
 +
<div class="menu-item">
 +
<span onclick="selectMenu(this.parentElement, 'Main Page')">
 +
<a href="https://2013.igem.org/Team:Tsinghua/Main-Page">Main Page</a>
 +
</span>
 +
</div>
 +
<div class="menu-item">
 +
<span onclick="selectMenu(this.parentElement, 'Introduction')">Introduction</span>
 +
<div class="sub-menu">
 +
<div class="sub-menu-item" onclick="selectSubMenu(this, 'Introduction - Background')">
 +
<a href="https://2013.igem.org/Team:Tsinghua/Introduction-Background">Background</a>
 +
</div>
 +
<div class="sub-menu-item" onclick="selectSubMenu(this, 'Introduction - Challenge')">
 +
<a href="https://2013.igem.org/Team:Tsinghua/Introduction-Challenge">Challenge</a>
 +
</div>
 +
<div class="sub-menu-item" onclick="selectSubMenu(this, 'Introduction - Our Idea')">
 +
<a href="https://2013.igem.org/Team:Tsinghua/Introduction-Our-Idea">Our Idea</a>
 +
</div>
 +
</div>
 +
</div>
 +
<div class="menu-item">
 +
<span onclick="selectMenu(this.parentElement, 'Project')">Project</span>
 +
<div class="sub-menu">
 +
<div class="sub-menu-item" onclick="selectSubMenu(this, 'Project - Overview')">
 +
<a href="https://2013.igem.org/Team:Tsinghua/Project-Overview">Overview</a>
 +
</div>
 +
<div class="sub-menu-item" onclick="selectSubMenu(this, 'Project - Part1: Sensor')">
 +
<a href="https://2013.igem.org/Team:Tsinghua/Project-Sensor">Part1: Sensor</a>
 +
</div>
 +
<div class="sub-menu-item" onclick="selectSubMenu(this, 'Project - Part2: Reporter')">
 +
<a href="https://2013.igem.org/Team:Tsinghua/Project-Reporter">Part2: Reporter</a>
 +
</div>
 +
<div class="sub-menu-item" onclick="selectSubMenu(this, 'Project - Part3: Switching System')">
 +
<a href="https://2013.igem.org/Team:Tsinghua/Project-Switching-System">Part3: Switching System</a>
 +
</div>
 +
<div class="sub-menu-item" onclick="selectSubMenu(this, 'Project - Product')">
 +
<a href="https://2013.igem.org/Team:Tsinghua/Project-Product">Product</a>
 +
</div>
 +
<div class="sub-menu-item" onclick="selectSubMenu(this, 'Project - Summary')">
 +
<a href="https://2013.igem.org/Team:Tsinghua/Project-Summary">Summary</a>
 +
</div>
 +
</div>
 +
</div>
 +
<div class="menu-item">
 +
<span onclick="selectMenu(this.parentElement, 'Modelling')">
 +
<a href="https://2013.igem.org/Team:Tsinghua/Modeling">Modeling</a>
 +
</span>
 +
</div>
 +
<div class="menu-item">
 +
<span onclick="selectMenu(this.parentElement, 'Outreach')">Outreach</span>
 +
<div class="sub-menu">
 +
<div class="sub-menu-item" onclick="selectSubMenu(this, 'Outreach - Human practice')">
 +
        Human practice
 +
      </div>
 +
<div class="sub-menu-item" onclick="selectSubMenu(this, 'Outreach - Safety')">
 +
<a href="https://2013.igem.org/Team:Tsinghua/OutReach-Satety">Safety</a>
 +
</div>
 +
<div class="sub-menu-item" onclick="selectSubMenu(this, 'Outreach - Collaboration')">
 +
<a href="https://2013.igem.org/Team:Tsinghua/OutReach-Collaboration">Collaboration</a>
 +
</div>
 +
</div>
 +
</div>
 +
<div class="menu-item">
 +
<span onclick="selectMenu(this.parentElement, 'Achievement')">Achievement</span>
 +
<div class="sub-menu">
 +
<div class="sub-menu-item" onclick="selectSubMenu(this, 'Achievement - BioBricks')">
 +
        BioBricks
 +
      </div>
 +
<div class="sub-menu-item" onclick="selectSubMenu(this, 'Achievement - Judging Criteria')">
 +
<a href="https://2013.igem.org/Team:Tsinghua/Achivement-Judging-Criteria">Judging Criteria</a>
 +
</div>
 +
</div>
 +
</div>
 +
<div class="menu-item">
 +
<span onclick="selectMenu(this.parentElement, 'Notebook')">Notebook</span>
 +
<div class="sub-menu">
 +
<div class="sub-menu-item" onclick="selectSubMenu(this, 'Notebook - Protocol')">
 +
<a href="https://2013.igem.org/Team:Tsinghua/Notebook-Protocol">Protocol</a>
 +
</div>
 +
<div class="sub-menu-item" onclick="selectSubMenu(this, 'Notebook - Lablog')">
 +
        Lablog
 +
      </div>
 +
</div>
 +
</div>
 +
<div class="menu-item">
 +
<span onclick="selectMenu(this.parentElement, 'Team')">Team</span>
 +
</div>
 +
<div class="menu-item">
 +
<span onclick="selectMenu(this.parentElement, 'Acknowledgement')">
 +
<a href="https://2013.igem.org/Team:Tsinghua/Acknowledgement">Acknowledgement</a><a>
 +
</a></span>
 +
</div>
 +
</div>
 +
</div>
 +
<div id="mycontent">
 +
<div class="normal">
 +
<h1>Protocol</h1>
 +
<h2>Standard Yeast Transformation Protocol</h2>
 +
<ol class="list">
 +
<li>
 +
    Inoculate a single colony of the desired yeast strain to be transformed into 5 ml YPD or selective media and grow overnight at the appropriate temperature.
 +
    </li>
 +
<li>
 +
    From the above overnight culture, inoculate into 5 ml YPD/selective media the appropriate amount to make the culture's optical density at 600 nm (OD600) equal to 0.1.
 +
    </li>
 +
<li>
 +
    Shake at the appropriate temperature until OD600 is between 0.6 and 1.0 (Normally I won’t let the OD reach over 1.0. Depends on strains, it may take 6-8 hours).
 +
    </li>
 +
<li>
 +
    Pellet cells 5 min at 3,000 rpm at room temperature.
 +
    </li>
 +
<li>
 +
    Wash the cells with 1ml ddH2O and transfer them into 1.5ml eppendorf tube. Pellet the cells at 3000rpm for 1min and remove the supernatant.
 +
    </li>
 +
<li>
 +
    Wash the cells with 1 ml 0.1M LioAc/TE buffer. Pellet cells and resuspend in 100ul of 0.1M LioAc/TE.
 +
    </li>
 +
<li>
 +
    Aliquot 20 ul of cell suspension into each of tube and add 2ul of plasmid DNA
 +
    </li>
 +
<li>
 +
    Add 80ul of transformation solution and mix well by pipetting up and down for several times
 +
    <p>
 +
      Transformation Solution (each):
 +
    </p>
 +
<p>
 +
      50% PEG  62.4ul
 +
    </p>
 +
<p>
 +
      1M LioAc      8.22ul
 +
    </p>
 +
<p>
 +
      DMSO      9.58ul
 +
    </p>
 +
<p>
 +
      10mg/ml ssDNA  5ul
 +
    </p>
 +
</li>
 +
<li>
 +
    Incubate the plate at 30°C for 30min (can be longer but not shorter than 30min)
 +
    </li>
 +
<li>
 +
    Heat shock by placing in a 42°C water bath for 15min
 +
    </li>
 +
<li>
 +
    Pellet the cells by centrifuging at 3000 rpm for 1min.
 +
    </li>
 +
<li>
 +
    Remove the supernatant.
 +
    </li>
 +
<li>
 +
    Wash the cells with 100ul of 5mM CaCl2. Pellet the cells (3000rpm, 1min) and remove the supernatant.
 +
    </li>
 +
<li>
 +
    Add 100ul of ddH2O, mix well on the plate shaker.
 +
    </li>
 +
<li>
 +
    Plate cells onto selective plates.
 +
    </li>
 +
</ol>
 +
<h2>Yeast Dry Powder Preparation Protocol</h2>
 +
<ol class="list">
 +
<li>
 +
    Inoculate a single colony of the desired yeast strain into 5 ml YPD media and grow overnight at the appropriate temperature. Determine OD600.
 +
    </li>
 +
<li>
 +
    Centrifuge at 3000 rpm for 5min. Remove the supernatant. Resuspend the cells in the media left. Transfer them into a 1.5ml eppendorf tube. Centrifuge at 3000 rpm for 1min.
 +
    </li>
 +
<li>
 +
    Remove the supernatant using pipette. Wash the cells with 120µl ddH2O.
 +
    </li>
 +
<li>
 +
    Centrifuge at 3000 rpm for 1min. Remove the supernatant.
 +
    </li>
 +
<li>
 +
    Dry in the vacuum centrifuge at 30℃ for 45min (or more).
 +
    </li>
 +
<li>
 +
    Grind the clotted yeast. Store in eppendorf tube at room temperature or -20℃.
 +
    </li>
 +
<li>
 +
    Test after one week.
 +
    </li>
 +
</ol>
 +
<h2>Western Blot Protocol</h2>
 +
<ol class="list">
 +
<li>
 +
    Sample Preparation
 +
    <p>Add 100µl 0.2M NaOH into 100µl yeast culture media. Incubate at room temperature for 5min. Centrifuge at 7000 rpm for 1min. Remove the supernatant.
 +
    </p>
 +
<p>
 +
    Resuspend the cells in 50µl SDS sample buffer. Incubate at 100℃ for 5min. Centrifuge at 7000 rpm for 1min. Keep the supernatant. Store at -20℃.
 +
    </p>
 +
</li>
 +
<li>
 +
    SDS-PAGE
 +
    <p>
 +
    The separation gel component is as below:
 +
    </p>
 +
<table border="1" class="center">
 +
<tr>
 +
<th>Percent(%)</th><th>7.5</th><th> 7.5 </th><th>10</th><th>10</th><th>12.5</th><th>12.5</th><th>15</th><th>15</th>
 +
</tr>
 +
<tr>
 +
<td></td><td>2</td><td>4</td><td>2</td><td>4</td><td>2</td><td>4</td><td>2</td><td>4</td>
 +
</tr>
 +
<tr>
 +
<td>1.5M Tris-HCl with SDS(PH 8.8) (ml)</td><td>2.5</td><td>5</td><td>2.5</td><td>5</td><td>2.5</td><td>5</td><td>2.5</td><td>5</td>
 +
</tr>
 +
<tr>
 +
<td>30% Acrylamide(ml)</td><td>2.5</td><td>5</td><td>3.4</td><td>6.8</td><td>4.2</td><td>8.4</td><td>5.0</td><td>10</td>
 +
</tr>
 +
<tr>
 +
<td>dd H2O (ml)</td><td>4.9</td><td>9.8</td><td>4.1</td><td>8.2</td><td>3.2</td><td>6.4</td><td>2.4</td><td>4.8</td>
 +
</tr>
 +
<tr>
 +
<td>10% APS (ul)</td><td>60</td><td>120</td><td>60</td><td>120</td><td>60</td><td>120</td><td>60</td><td>120</td>
 +
</tr>
 +
<tr>
 +
<td>TEMED (ul)</td><td>6</td><td>12</td><td>6</td><td>12</td><td>6</td><td>12</td><td>6</td><td>12</td>
 +
</tr>
 +
<tr>
 +
<td>Total Volum(ml)</td><td>10</td><td>20</td><td>10</td><td>20</td><td>10</td><td>20</td><td>10</td><td>20</td>
 +
</tr>
 +
</table>
 +
<p>stacking gel component is as below:</p>
 +
<table border="1" class="center">
 +
<tr>
 +
<td></td><td>2</td><td>4</td>
 +
</tr>
 +
<tr>
 +
<td>1.5M Tris-HCl with SDS(PH 8.8) (ml)</td><td>1.25</td><td>2.5</td>
 +
</tr>
 +
<tr>
 +
<td>30% Acrylamide(ml)</td><td>0.7</td><td>1.4</td>
 +
</tr>
 +
<tr>
 +
<td>dd H2O (ml)</td><td>3</td><td>6</td>
 +
</tr>
 +
<tr>
 +
<td>10% APS (ul)</td><td>30</td><td>60</td>
 +
</tr>
 +
<tr>
 +
<td>TEMED (ul)</td><td>6</td><td>12</td>
 +
</tr>
 +
<tr>
 +
<td>Total Volum(ml)</td><td>5</td><td>10</td>
 +
</tr>
 +
</table>
 +
<p>
 +
    Add protein sample to each well.
 +
    </p>
 +
<p>
 +
    Then use 100V to run. When the proteins reach the boundary of separation and stacking gel, you can change the voltage into 140V. Stop when the proteins reach the bottom of gel.
 +
    </p>
 +
</li>
 +
<li>
 +
    Blocking and Antibody Incubation
 +
    <p>
 +
      Use the 5% milk blocking buffer to block the NC membrane at room temperature for 1h.
 +
    </p>
 +
<p>
 +
      Transfer the NC membrane to first antibody buffer (rabbit anti-flag 1:4000), at 4℃ overnight.
 +
    </p>
 +
<p>
 +
      Use the PBST to wash the NC membrane for 3 times, each for 10min at least.
 +
    </p>
 +
<p>
 +
      Then transfer the NC membrane to the second antibody buffer (rabbit anti-IgG 1:10000). Incubate at room temperature for 1h.
 +
    </p>
 +
<p>
 +
      Use the PBST to wash the NC membrane for 3 times, each for 10min at least.
 +
    </p>
 +
</li>
 +
<li>
 +
    Development
 +
    <p>
 +
      Add A solution and B solution (1:1), and mix them in the plate.
 +
    </p>
 +
<p>
 +
      Put the membrane into the plate and let the liquid flow past the NC membrane. Take picture of your western result.
 +
    </p>
 +
</li>
 +
</ol>
 +
<h2>Constructing Insert into Vector</h2>
 +
<p>The method we used in our experiment is typical molecular cloning approach.
 +
  </p>
 +
<div class="figure">
 +
<img class="center" src="https://static.igem.org/mediawiki/2013/1/18/Tsinghua-protocol1.png"/>
 +
<p class="legend">
 +
    Figure 1.
 +
    </p>
 +
</div>
 +
<ol class="list">
 +
<li>
 +
      Design primers.
 +
    </li>
 +
<li>
 +
    Amplify DNA of target site with specific enzyme digestion site.
 +
    </li>
 +
<li>
 +
    Digest insert and vector separately.
 +
    </li>
 +
<li>
 +
    Perform ligation.
 +
    </li>
 +
<li>
 +
    Select positive clones.
 +
    </li>
 +
</ol>
 +
<h2>AHL induction and flow cytometry analysis</h2>
 +
<p>
 +
    The response of engineered yeast to AHL quorum sensing signals was examined using pTF4 transformants, where AHL signals would be recognized by LuxR receptors, thereby promoting mCherry fluorescent protein expression. Induced fluorescence was checked using flow cytometry analysis. Samples were prepared as specified below.
 +
  </p>
 +
<ol class="list">
 +
<li>
 +
    pTF4 transformant yeast was cultured in SC-His selective medium from 0.1 OD for 8 hours, at 30 ℃, shaking culture.
 +
    </li>
 +
<li>
 +
    Yeast cells were induced with AHL.
 +
    <p>
 +
    a. 0.5 μM AHL induction:  Added 25 μL AHL (0.1 mM stock solution) into 5 mL pTF4 transformant yeast culture.
 +
    </p>
 +
<p>
 +
    b. Ctrl: Added 25 μL ddH2O as control.
 +
    </p>
 +
<p>30℃ shaking culture.</p>
 +
</li>
 +
<li>
 +
    Sample fixation: 0.5h, 2h, 8h and 24h post-induction: Collect 500 μM sample and fix in 10% formalin for 15 min. Formalin was removed after fixation and yeast cells were resuspended in 500 μM PBS and stored at 4℃.
 +
    </li>
 +
<li>
 +
    Samples were subjected to flow cytometry analysis to check for mCherry fluorescence. Negative control was established using native yeast with no mCherry expression.
 +
    </li>
 +
</ol>
 +
</div>
 +
</div>
 +
</div>
 +
</body></html>

Revision as of 04:10, 26 September 2013

Protocol

Standard Yeast Transformation Protocol

  1. Inoculate a single colony of the desired yeast strain to be transformed into 5 ml YPD or selective media and grow overnight at the appropriate temperature.
  2. From the above overnight culture, inoculate into 5 ml YPD/selective media the appropriate amount to make the culture's optical density at 600 nm (OD600) equal to 0.1.
  3. Shake at the appropriate temperature until OD600 is between 0.6 and 1.0 (Normally I won’t let the OD reach over 1.0. Depends on strains, it may take 6-8 hours).
  4. Pellet cells 5 min at 3,000 rpm at room temperature.
  5. Wash the cells with 1ml ddH2O and transfer them into 1.5ml eppendorf tube. Pellet the cells at 3000rpm for 1min and remove the supernatant.
  6. Wash the cells with 1 ml 0.1M LioAc/TE buffer. Pellet cells and resuspend in 100ul of 0.1M LioAc/TE.
  7. Aliquot 20 ul of cell suspension into each of tube and add 2ul of plasmid DNA
  8. Add 80ul of transformation solution and mix well by pipetting up and down for several times

    Transformation Solution (each):

    50% PEG 62.4ul

    1M LioAc 8.22ul

    DMSO 9.58ul

    10mg/ml ssDNA 5ul

  9. Incubate the plate at 30°C for 30min (can be longer but not shorter than 30min)
  10. Heat shock by placing in a 42°C water bath for 15min
  11. Pellet the cells by centrifuging at 3000 rpm for 1min.
  12. Remove the supernatant.
  13. Wash the cells with 100ul of 5mM CaCl2. Pellet the cells (3000rpm, 1min) and remove the supernatant.
  14. Add 100ul of ddH2O, mix well on the plate shaker.
  15. Plate cells onto selective plates.

Yeast Dry Powder Preparation Protocol

  1. Inoculate a single colony of the desired yeast strain into 5 ml YPD media and grow overnight at the appropriate temperature. Determine OD600.
  2. Centrifuge at 3000 rpm for 5min. Remove the supernatant. Resuspend the cells in the media left. Transfer them into a 1.5ml eppendorf tube. Centrifuge at 3000 rpm for 1min.
  3. Remove the supernatant using pipette. Wash the cells with 120µl ddH2O.
  4. Centrifuge at 3000 rpm for 1min. Remove the supernatant.
  5. Dry in the vacuum centrifuge at 30℃ for 45min (or more).
  6. Grind the clotted yeast. Store in eppendorf tube at room temperature or -20℃.
  7. Test after one week.

Western Blot Protocol

  1. Sample Preparation

    Add 100µl 0.2M NaOH into 100µl yeast culture media. Incubate at room temperature for 5min. Centrifuge at 7000 rpm for 1min. Remove the supernatant.

    Resuspend the cells in 50µl SDS sample buffer. Incubate at 100℃ for 5min. Centrifuge at 7000 rpm for 1min. Keep the supernatant. Store at -20℃.

  2. SDS-PAGE

    The separation gel component is as below:

    Percent(%)7.5 7.5 101012.512.51515
    24242424
    1.5M Tris-HCl with SDS(PH 8.8) (ml)2.552.552.552.55
    30% Acrylamide(ml)2.553.46.84.28.45.010
    dd H2O (ml)4.99.84.18.23.26.42.44.8
    10% APS (ul)60120601206012060120
    TEMED (ul)612612612612
    Total Volum(ml)1020102010201020

    stacking gel component is as below:

    24
    1.5M Tris-HCl with SDS(PH 8.8) (ml)1.252.5
    30% Acrylamide(ml)0.71.4
    dd H2O (ml)36
    10% APS (ul)3060
    TEMED (ul)612
    Total Volum(ml)510

    Add protein sample to each well.

    Then use 100V to run. When the proteins reach the boundary of separation and stacking gel, you can change the voltage into 140V. Stop when the proteins reach the bottom of gel.

  3. Blocking and Antibody Incubation

    Use the 5% milk blocking buffer to block the NC membrane at room temperature for 1h.

    Transfer the NC membrane to first antibody buffer (rabbit anti-flag 1:4000), at 4℃ overnight.

    Use the PBST to wash the NC membrane for 3 times, each for 10min at least.

    Then transfer the NC membrane to the second antibody buffer (rabbit anti-IgG 1:10000). Incubate at room temperature for 1h.

    Use the PBST to wash the NC membrane for 3 times, each for 10min at least.

  4. Development

    Add A solution and B solution (1:1), and mix them in the plate.

    Put the membrane into the plate and let the liquid flow past the NC membrane. Take picture of your western result.

Constructing Insert into Vector

The method we used in our experiment is typical molecular cloning approach.

Figure 1.

  1. Design primers.
  2. Amplify DNA of target site with specific enzyme digestion site.
  3. Digest insert and vector separately.
  4. Perform ligation.
  5. Select positive clones.

AHL induction and flow cytometry analysis

The response of engineered yeast to AHL quorum sensing signals was examined using pTF4 transformants, where AHL signals would be recognized by LuxR receptors, thereby promoting mCherry fluorescent protein expression. Induced fluorescence was checked using flow cytometry analysis. Samples were prepared as specified below.

  1. pTF4 transformant yeast was cultured in SC-His selective medium from 0.1 OD for 8 hours, at 30 ℃, shaking culture.
  2. Yeast cells were induced with AHL.

    a. 0.5 μM AHL induction: Added 25 μL AHL (0.1 mM stock solution) into 5 mL pTF4 transformant yeast culture.

    b. Ctrl: Added 25 μL ddH2O as control.

    30℃ shaking culture.

  3. Sample fixation: 0.5h, 2h, 8h and 24h post-induction: Collect 500 μM sample and fix in 10% formalin for 15 min. Formalin was removed after fixation and yeast cells were resuspended in 500 μM PBS and stored at 4℃.
  4. Samples were subjected to flow cytometry analysis to check for mCherry fluorescence. Negative control was established using native yeast with no mCherry expression.