Team:Warsaw/Calendar-Main/18 June 2008

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<h3>Preparation of constructs: OmpA_alpha and OmpA_omega</h3>
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<h3>Preparation of constructs with OmpA protein fusions</h3>
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<h4>Michał K.</h4>
<h4>Michał K.</h4>
<ol><li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177>pACYC177</a>.</li>
<ol><li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177>pACYC177</a>.</li>
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<li>Plating transformants on LB+tetracycline.</li></ol>  
<li>Plating transformants on LB+tetracycline.</li></ol>  
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<a name="fig1"> <img src="https://static.igem.org/mediawiki/2008/c/c9/Ompa_Alpha_Omega_Cleanup_WAW.jpg" width=300/> </a>
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<var> <b>Fig. 1. </b>OmpA_alpha (lane 2) and OmpA_omega (lane 3) digest products.</var>
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<a name="fig2"><img src="https://static.igem.org/mediawiki/2008/5/5f/PACYC177_digest_WAW.jpg" width=300/></a>
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<var><b>Fig. 2.</b> pACYC177 digested with NdeI and BamHI (lane 2).</var>
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<h3> Blue/white and rifampicin test </h3>
 
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<h4>Michał L., Ewa, Marcin</h4>
 
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<h3> Blue/white and rifampicin test #2</h3>
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<h4>Michał L., Ewa, Piotr</h4>
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<p>Counting of blue colonies: </p>
<p>Counting of blue colonies: </p>
<table id="result" align="left">
<table id="result" align="left">
<tr><th>Strain</th><th>% of blue colonies</th></tr>
<tr><th>Strain</th><th>% of blue colonies</th></tr>
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<tr><td>pMPMT5</td><td>99%</td></tr>
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<tr><td>pMPMT5</td><td>96%</td></tr>
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<tr><td>pMPMT5-AID</td><td>93%</td></tr>
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<tr><td>pMPMT5-AID</td><td>98%</td></tr>
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<tr><td>pMPMT5-AIDT7</td><td>81%</td></tr>
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<tr><td>pMPMT5-AIDT7</td><td>61%</td></tr>
<tr><td>pMPMT5-AID+T7</td><td>1%</td></tr>
<tr><td>pMPMT5-AID+T7</td><td>1%</td></tr>
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<table id="result">
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<thead><b> Results of rifampicin test</b><br></thead>
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<tr><th>Strain</th><th>Inducer / repressor</th><th>Number of colonies on rifampicin</th><th>OD</th></tr>
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<tr><th>Top10</th><td>none</td><td>3</td><th>2.91</th></tr>
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<tr><th>Top10 pMPM-AID</th><td>0.1% Arabinose</td><td>72</td><th>2.7</tr>
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<tr><th>Top10 pMPM-AID</th><td>none</td><td>8</td><th>2.58</tr>
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<tr><th>Top10 pMPM-AID+T7</th><td>0.1% Arabinose</td><td>148</td><th>1.88</th></tr>
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<tr><th>Top10 pMPM-AID+T7</th><td>none</td><td>6</td><th>2.52</th></tr>
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<tr><th>Top10 pMPM-AIDT7</th><td>0.1% Arabinose</td><td>17</td><th>2.74</th></tr>
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<tr><th>Top10 pMPM-AIDT7</th><td>none</td><td>21</td><th>2.59</th></tr>
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<p>AID in transcription fusion works like AID, but induce slower growth than AID;<br>
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AID in translation fusion do not induce rifampicin resistance mutation
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<table id="result" align="left">
 
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<tr><th colspan="3"> Results of rifampicin test</th></tr>
 
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<tr><th>Strain</th><th>Inducer / repressor</th><th>Number of colonies on rifampicin</th></tr>
 
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<tr><th>Top10</th><td>none</td><td>8</td></tr>
 
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<tr><th>Top10 pMPM-AID</th><td>0.1% Arabinose</td><td>72</td></tr>
 
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<tr><th>Top10 pMPM-AID</th><td>none</td><td>4</td></tr>
 
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<tr><th>Top10 pMPM-A+T13</th><td>0.1% Arabinose</td><td>148</td></tr>
 
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<tr><th>Top10 pMPM-A+T13</th><td>none</td><td>6</td></tr>
 
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<tr><th>Top10 pMPM-AT6</th><td>0.1% Arabinose</td><td>21</td></tr>
 
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<tr><th>Top10 pMPM-AT6</th><td>none</td><td>27</td></tr>
 
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<a name="fig1"> <img src="https://static.igem.org/mediawiki/2008/c/c9/Ompa_Alpha_Omega_Cleanup_WAW.jpg" width=300/> </a>
 
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<var> <b>Fig. 1. </b>OmpA_alpha (lane 2) and OmpA_omega (lane 3) digest products.</var>
 
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<a name="fig2"><img src="https://static.igem.org/mediawiki/2008/5/5f/PACYC177_digest_WAW.jpg" width=300/></a>
 
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<var><b>Fig. 2.</b> pACYC177 digested with NdeI and BamHI (lane 2).</var>
 
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Latest revision as of 14:55, 26 October 2008

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Preparation of constructs: OmpA_alpha and OmpA_omega

Michał K.

  1. Isolation of pACYC177.
  2. Digest of purified pACYC177 with NdeI and BamHI (Tango 2x buffer).
  3. Gel electrophoresis (Fig. 2) and isolation of proper band (3200 bp).
  4. Clean-up of OmpA_alpha and OmpA_omega digest products (Fig. 1).
  5. Ligation of purified DNA (pACYC177 with OmpA_alpha and OmpA_omega DNA fragments).
  6. Transformation of E.coli TOP10 with ligation products.
  7. Plating transformants on LB+tetracycline.
Fig. 1. OmpA_alpha (lane 2) and OmpA_omega (lane 3) digest products. Fig. 2. pACYC177 digested with NdeI and BamHI (lane 2).

Blue/white and rifampicin test #2

Michał L., Ewa, Piotr

Counting of blue colonies:

Strain% of blue colonies
pMPMT596%
pMPMT5-AID98%
pMPMT5-AIDT761%
pMPMT5-AID+T71%









Results of rifampicin test
StrainInducer / repressorNumber of colonies on rifampicinOD
Top10none32.91
Top10 pMPM-AID0.1% Arabinose722.7
Top10 pMPM-AIDnone82.58
Top10 pMPM-AID+T70.1% Arabinose1481.88
Top10 pMPM-AID+T7none62.52
Top10 pMPM-AIDT70.1% Arabinose172.74
Top10 pMPM-AIDT7none212.59

AID in transcription fusion works like AID, but induce slower growth than AID;
AID in translation fusion do not induce rifampicin resistance mutation

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