Team:Warsaw/Calendar-Main/21 August 2008

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<h3>Tests for ampicillin resistance given by protein added to medium</h3><h4>Piotr</h4>
<h3>Tests for ampicillin resistance given by protein added to medium</h3><h4>Piotr</h4>
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Results of growth from previus day
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Results of growth from previous day
<br><br>
<br><br>
<table id="result">
<table id="result">
<tr><th>OmpA variant ("hunter")</th><th>"Prey" variant</th><th>Growth with prey</th><th>Growth without prey</th></tr>
<tr><th>OmpA variant ("hunter")</th><th>"Prey" variant</th><th>Growth with prey</th><th>Growth without prey</th></tr>
<tr><th>OmpA-alpha</th><td>His+Z+omega</td><td>+</td><td>+</td></tr>
<tr><th>OmpA-alpha</th><td>His+Z+omega</td><td>+</td><td>+</td></tr>
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<tr><th>OmpA-alpha</th><td>His+Z+alfa</td><td>+</td><td>+</td></tr>
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<tr><th>OmpA-alpha</th><td>His+Z+alpha</td><td>+</td><td>+</td></tr>
<tr><th>OmpA-A-alpha</th><td>His+Z+omega</td><td>+</td><td>-</td></tr>
<tr><th>OmpA-A-alpha</th><td>His+Z+omega</td><td>+</td><td>-</td></tr>
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<tr><th>OmpA-Z-omega</th><td>His+A+alpha</td><td>+</td><td>-</td></tr>
 
<tr><th>Ompa-A-omega</th><td>His+Z+alpha</td><td>+</td><td>-</td></tr>
<tr><th>Ompa-A-omega</th><td>His+Z+alpha</td><td>+</td><td>-</td></tr>
<tr><th>OmpA-omega</th><td>His+Z+alpha</td><td>+</th><td>-</td></tr>
<tr><th>OmpA-omega</th><td>His+Z+alpha</td><td>+</th><td>-</td></tr>
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</table>
</table>
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<br><br>
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<ol><li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation of plasmids</a> from above cultures. </li>
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<li> Control
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digest</a> of isolated plasmids with BamHI and SacI.</li> <li> Gel electrophoresis - all constructs apart from <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha>pACYC177+OmpA_alpha</a> were successfully recovered.</li>
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</ol></p>
<br>
<br>
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OmpA-alpha may be comtaminated with antibiotic resistance bacteria, alpha do not need interaction of A and Z to bind to omega - maybe we must give bacteria less pray
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<p><b>Conclusions:</b>
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<ol>
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<li>Almost none of tested constructs give resistance to ampicillin without prey protein added to culture medium.</li>
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<li>Interaction between hunter and prey is not necessary to make cells ampicillin resistant (OmpA_alpha and OmpA_omega grow with prey).</li>
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<li><a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha>pACYC177+OmpA_alpha</a> culture may be contaminated with antibiotic-resistant bacteria, alpha doesn't need interaction of A and Z proteins to bind to omega - maybe we must give bacteria less prey.</li></ol></p>
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<html>
 
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<br>
 
<h3>Cloning of protein A DNA to GeneArt plasmid</h3><h4>Antoni</h4>
<h3>Cloning of protein A DNA to GeneArt plasmid</h3><h4>Antoni</h4>
<p><ol>
<p><ol>
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL+SacI">AL+SacI</a>. </li>  
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL+SacI">AL+SacI</a>. </li>  
<li>Gel electrophoresis.</li>
<li>Gel electrophoresis.</li>
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<li> Confirmed transformant colonies inoculated to liquid LB with ampicillin.
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<li> Confirmed transformant colonies inoculated to liquid LB with ampicillin.</li>
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</li>
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<li>Inoculation to liquid LB with ampicillin: <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-alpha>pET15b+OmpA-alpha</a>.</li>
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</ol></p>
</ol></p>
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<h3>Cloning of alpha to <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-A-omega>pACYC177+OmpA_A_omega</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-Z-omega>pACYC177+OmpA_Z_omega</a></h3><h4>Michał K.</h4>
<h3>Cloning of alpha to <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-A-omega>pACYC177+OmpA_A_omega</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-Z-omega>pACYC177+OmpA_Z_omega</a></h3><h4>Michał K.</h4>
<p><ol>
<p><ol>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation of plasmids</a> from cultures inocluated on previous day. </li>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation of plasmids</a> from cultures inoculated on previous day. </li>
<li> Control  
<li> Control  
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digest</a> of isolated plasmids with FastBamHI and FastAcc65I (Fast Digest buffer).</li> <li> Gel electrophoresis - no proper clones founded.</li>
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digest</a> of isolated plasmids with FastBamHI and FastAcc65I (Fast Digest buffer).</li>  
 +
<li> Gel electrophoresis - no proper clones found (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/21_August_2008#fig1">Fig. 1.</a>).</li>
</ol></p>
</ol></p>
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 +
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/f/f3/Traw_04_09_2008.jpg" width=300/></a> <var><b>Fig. 1.</b> Control Acc65I/BamHI digests of isolated plasmids<br>
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1. Marker<br>
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2-3. digested plasmids pACYC177+OmpA_A_omega+alpha <br>
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4-5. digested plasmids pACYC177+OmpA_Z_omega+alpha <br></var>
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Tests for ampicillin resistance given by protein added to medium

Piotr

Results of growth from previous day

OmpA variant ("hunter")"Prey" variantGrowth with preyGrowth without prey
OmpA-alphaHis+Z+omega++
OmpA-alphaHis+Z+alpha++
OmpA-A-alphaHis+Z+omega+-
Ompa-A-omegaHis+Z+alpha+-
OmpA-omegaHis+Z+alpha+-
Ompa-Z-alphaHis+Z+omega+-
OmpA_Adelta_alphaHis+Z+omega+-
OmpA_Adelta_omegaHis+Z+alpha+-
OmpA_omega_AdeltaHis+Z+alpha+-
OmpA-omega-A-alphano++


  1. Isolation of plasmids from above cultures.
  2. Control digest of isolated plasmids with BamHI and SacI.
  3. Gel electrophoresis - all constructs apart from pACYC177+OmpA_alpha were successfully recovered.


Conclusions:

  1. Almost none of tested constructs give resistance to ampicillin without prey protein added to culture medium.
  2. Interaction between hunter and prey is not necessary to make cells ampicillin resistant (OmpA_alpha and OmpA_omega grow with prey).
  3. pACYC177+OmpA_alpha culture may be contaminated with antibiotic-resistant bacteria, alpha doesn't need interaction of A and Z proteins to bind to omega - maybe we must give bacteria less prey.

Cloning of protein A DNA to GeneArt plasmid

Antoni

  1. Colony PCR on colonies from plates with transformations pGeneart+A.
    Primers used: AP+NotI and AL+SacI.
  2. Gel electrophoresis.
  3. Confirmed transformant colonies inoculated to liquid LB with ampicillin.

Cloning of alpha to pACYC177+OmpA_A_omega and pACYC177+OmpA_Z_omega

Michał K.

  1. Isolation of plasmids from cultures inoculated on previous day.
  2. Control digest of isolated plasmids with FastBamHI and FastAcc65I (Fast Digest buffer).
  3. Gel electrophoresis - no proper clones found (Fig. 1.).

Fig. 1. Control Acc65I/BamHI digests of isolated plasmids
1. Marker
2-3. digested plasmids pACYC177+OmpA_A_omega+alpha
4-5. digested plasmids pACYC177+OmpA_Z_omega+alpha


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