Team:Warsaw/Calendar-Main/30 September 2008

Mutagenesis of protein A Paweł

Results of sequencing: unfortunately all sequences were wild-type.

Preparation of vectors for Biobricks Michał K., Piotr  Isolation of pSB2K3 and BBa_I739204 (pACYC177 converted into BioBrick vector) plasmids.

Digest of isolated plasmids with EcoRI and BcuI (BamHI buffer). Dephosphorylation (CIAP) of plasmids.

Inoculation of bacteria received from iGEM HQs, carrying pSB2K3</a> and BBa_I739204 (pACYC177 converted into BioBrick vector)</a> plasmids.</li> Gel electrophoresis and gel-out</a> of proper bands: 4500 bp - pSB2K3</a> and 3000 bp - BBa_I739204 (pACYC177 converted into BioBrick vector)</a>. Fig. 1</a>. </li>

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<img src="http://2008.igem.org/wiki/images/7/79/Go_30_09_2008.jpg " width=200/></a> </a> Fig. 1. EcoRI/BcuI digests of isolated plasmids 1. Marker 2-3. Digested plasmids BBa_I739204 4. Digested plasmid psB2K3

Preparation of OmpA-linker-omega-linker (BBa_K103016)</a> Michał K.

<ol> Digest</a> of OmpA-linker-omega-linker (BBa_K103016)</a> PCR product with BglII and PstI (Orange buffer). </li> Clean-up</a> of above digest reaction. </li>  Digest</a> of pACYC177+OmpA_omega_deltaA_alpha</a> with BamHI and PstI (BamHI buffer). <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">Dephosphorylation</a> (CIAP) of plasmid.</li>

<li>Gel electrophoresis and <a href="http://2008.igem.org/Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper band - 3800 bp. <a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/30_September_2008#fig2">Fig. 2</a>. </li> </ol>

<img src="http://2008.igem.org/wiki/images/8/8f/Go2_30_09_2008na30_09.jpg" width=150/></a> </a> Fig. 2. BamHI/PstI digests of isolated plasmid 1. Marker 2. Digested plasmid pACYC_OmpA_omega_deltaA_alpha

Preparation of <a href=http://partsregistry.org/Part:BBa_K103006>OmpA-linker (BBa_K103006)</a> Piotr <ol><li> E. coli <a href="http://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">transformation</a> with overnight ligation <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a>+<a href=http://partsregistry.org/Part:BBa_K103006>OmpA-linker (BBa_K103006)</a>. </li>

<li>Plating on LB + ampicillin.</li></ol>

Preparation of <a href=http://partsregistry.org/Part:BBa_K103020>omega_linker under PT7 (BBa_K103020)</a> Michał K. <ol> <li><a href="http://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of omega_linker fragment (PCR from previous day) with NdeI and SacI (BamHI buffer).</li>

<li><a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_purification_after_enzymatic_reaction">Clean-up</a> of above digest reaction. </li></ol>

Preparation of <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103004>Z(BBa_K103004)</a> Piotr <ol><li> E. coli <a href="http://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">transformation</a> with overnight ligations <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> + <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103004>Z(BBa_K103004)</a>. </li>

<li>Plating on LB + ampicillin.</li></ol>

Preparation of <a href=http://partsregistry.org/Part:BBa_K103018>OmpA_linker_omega_linker under Plac (BBa_K103018)</a> Michał K.

Overnight <a href="http://2008.igem.org/Wiki/Team:Warsaw/protocols#digest">digest</a> of purified <a href=http://partsregistry.org/Part:BBa_K103018>BBa_K103018</a> fragment (from previous day) with EcoRI (EcoRI buffer). DNA ends <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#blunting">blunting</a> with Klenow fragment.