Team:Warsaw/Calendar-Main/21 August 2008

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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>
<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  
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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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<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>
</ol></p>
</ol></p>
<br>
<br>
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<ol>
<ol>
<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>
-
<li>Interaction between hunter and prey is not necessary to make cells ampicillin resistant (OmpA_alpha and OmpA_omega grow with prey)</li>
+
<li>Interaction between hunter and prey is not necessary to make cells ampicillin resistant (OmpA_alpha and OmpA_omega grow with prey).</li>
<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>
<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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<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> <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  
-
<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) <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/21_August_2008#fig1">Fig. 1</a>.</li> <li> Gel electrophoresis - no proper clones found.</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 (<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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<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. Control Acc65I/BamHI digests of isolated plasmids</b><br>  
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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>  
1. Marker<br>
1. Marker<br>
2-3. digested plasmids pACYC177+OmpA_A_omega+alpha <br>
2-3. digested plasmids pACYC177+OmpA_A_omega+alpha <br>

Latest revision as of 19:09, 26 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+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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