Team:Warsaw/Calendar-Main/21 August 2008

From 2008.igem.org

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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 abow cultures. </li>
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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>
<li> Control  
<li> Control  
<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 ompa_alfa were successfully recovered</li>
<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 ompa_alfa were successfully recovered</li>
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<li>Almost none of tested constructs give resistance to ampicillin without prey protein added to culture medium.</li>
<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 nessesery to makes cells ampicillin resistant(Omp_alpha and Omp_omega growth with prey)</li>
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<li>Interaction between hunter and prey is not necessary to makes cells ampicillin resistant(Omp_alpha and Omp_omega growth with prey)</li>
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<li>OmpA-alpha may be comtaminated with antibiotic-resistant bacteria, alpha do not need interaction of A and Z to bind to omega - maybe we must give bacteria less prey</li>
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<li>OmpA-alpha may be contaminated with antibiotic-resistant bacteria, alpha do not need interaction of A and Z to bind to omega - maybe we must give bacteria less prey</li>
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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>
+
<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>
+
<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.</li>
</ol></p>
</ol></p>

Revision as of 15:59, 24 October 2008

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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+alfa++
OmpA-A-alphaHis+Z+omega+-
OmpA-Z-omegaHis+A+alpha+-
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 ompa_alfa 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 makes cells ampicillin resistant(Omp_alpha and Omp_omega growth with prey)
  3. OmpA-alpha may be contaminated with antibiotic-resistant bacteria, alpha do not need interaction of A and Z to bind to omega - maybe we must give bacteria less prey

    4. 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.
    4. Inoculation to liquid LB with ampicillin: pET15b+OmpA-alpha.

    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.

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