Team:Warsaw/Calendar-Main/18 June 2008

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<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of purified pACYC177 with NdeI and BamHI (Tango 2x buffer). </li>  
<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of purified pACYC177 with NdeI and BamHI (Tango 2x buffer). </li>  
<li> Gel electrophoresis and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_purification_after_enzymatic_reaction">isolation</a> of proper band (3200 bp).</li>
<li> Gel electrophoresis and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_purification_after_enzymatic_reaction">isolation</a> of proper band (3200 bp).</li>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">Clean-up</a> of OmpA_alpha and OmpA_omega digest products. </li>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">Clean-up</a> of OmpA_alpha and OmpA_omega digest products (<a href="https://2008.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/18_June_2008#fig1">Fig. 1</a>). </li>
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of purified DNA (pACYC177 with OmpA_alpha and OmpA_omega DNA fragments).</li>
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of purified DNA (pACYC177 with OmpA_alpha and OmpA_omega DNA fragments).</li>
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">Transformation</a> of <i>E.coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> with ligation products.</li>
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">Transformation</a> of <i>E.coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> with ligation products.</li>
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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> Fig. 1. OmpA_alpha (lane 2) and OmpA_omega(lane 3) digest products.</var>
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Revision as of 00:48, 12 October 2008

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Preparation of constructs with OmpA protein fusions

Michał K.

  1. Isolation of pACYC177.
  2. Digest of purified pACYC177 with NdeI and BamHI (Tango 2x buffer).
  3. Gel electrophoresis 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.

Blue/white and rifampicin test

Michał L., Ewa, Marcin

Counting of blue colonies:

Strain% of blue colonies
pMPMT599%
pMPMT5-AID93%
pMPMT5-AIDT781%
pMPMT5-AID+T71%

Results of rifampicin test
StrainInducer / repressorNumber of colonies on rifampicin
Top10none8
Top10 pMPM-AID0.1% Arabinose72
Top10 pMPM-AIDnone4
Top10 pMPM-A+T130.1% Arabinose148
Top10 pMPM-A+T13none6
Top10 pMPM-AT60.1% Arabinose21
Top10 pMPM-AT6none27
Fig. 1. OmpA_alpha (lane 2) and OmpA_omega(lane 3) digest products.