Team:Warsaw/Calendar-Main/27 June 2008

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<h3>Preparation of constructs with OmpA protein fusions</h3><h4>Michał K.</h4>
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<h3>Change of the reporter from <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pZC320>pZC320</a> with B-galactosidase to GFP or RFP</h3>
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<h4>Piotr, Weronika</h4>
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<li>Isolation of <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A2>pSB1A2</a> standard plasmids carrying parts: <A href=http://partsregistry.org/wiki/index.php?title=Part:BBa_E0840>BBa_E0840</a>(GFP genetrator) and <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_J04450>BBa_J04450</a> (RFP generator).</li>
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<li>Digest of <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A2>pSB1A2</a> standard plasmids carrying parts: <A href=http://partsregistry.org/wiki/index.php?title=Part:BBa_E0840>BBa_E0840</a>(GFP genetrator) and <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_J04450>BBa_J04450</a> (RFP generator) with NotI.</li>
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Digest of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pZC320>pZC320</a> with NotI and <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing>dephosphorylation</a> with CIAP.
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Gel electrophoresis and gel-out of proper bands.
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Ligation of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pZC320>pZC320</a> with standard parts: <A href=http://partsregistry.org/wiki/index.php?title=Part:BBa_E0840>BBa_E0840</a>(GFP genetrator) and <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_J04450>BBa_J04450</a> (RFP generator).
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<li>Chemotransformation of E.coli TOP10 with ligation products.</li>
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<li>Plating transformants on LB+Amp30+X-gal+IPTG.
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<h3>Preparation of constructs: OmpA_alpha and OmpA_omega</h3><h4>Michał K.</h4>
<p><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. </p>
<p><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. </p>
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<h3>Cloning PCL products on pKS</h3>
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<h3>Cloning alpha-A and omega-A fusions on <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pKSII%2B>pKS</a></h3>
<h4>Michał L., Ewa, Marcin</h4>
<h4>Michał L., Ewa, Marcin</h4>
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<li>Gel electophoresis of PCL products (Fig. 1.).</li>
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<li>Gel electophoresis of PCL products (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/27_June_2008#fig1">Fig. 1</a>).</li>
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<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">Gel-out</a> of proper bands (alpha-A: 1500 bp and omega-A: 1440 bp).</li>
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<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">Gel-out</a> of proper bands (alpha-A: 1100 bp and omega-A: 750 bp).</li>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">Restriction digest</a> of pKS vector and the PCL products with NotI and SacI (BamHI buffer) for 2 hours.</li>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">Restriction digest</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pKSII%2B>pKS</a> vector and the PCL products with NotI and SacI (BamHI buffer) for 2 hours.</li>
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> 1 hour.</li>
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> 1 hour.</li>
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">Transformation</a> of Top10 strain and screening on Amp<sub>100</sub> plates.</li>
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">Transformation</a> of Top10 strain and screening on Amp<sub>100</sub> plates.</li>
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<img src="https://static.igem.org/mediawiki/2008/e/ea/PCL_OmegaA_WAW.jpg" width=200/>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/e/ea/PCL_OmegaA_WAW.jpg" width=200/></a>
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<var><b>Fig. 1.</b>PCL product - Omega-A fusion.</var>
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<var><b>Fig. 1.</b>PCL product - Alpha-A fusion.</var>
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Change of the reporter from pZC320 with B-galactosidase to GFP or RFP

Piotr, Weronika

  1. Isolation of pSB1A2 standard plasmids carrying parts: BBa_E0840(GFP genetrator) and BBa_J04450 (RFP generator).
  2. Digest of pSB1A2 standard plasmids carrying parts: BBa_E0840(GFP genetrator) and BBa_J04450 (RFP generator) with NotI.
  3. Digest of pZC320 with NotI and dephosphorylation with CIAP.
  4. Gel electrophoresis and gel-out of proper bands.
  5. Ligation of pZC320 with standard parts: BBa_E0840(GFP genetrator) and BBa_J04450 (RFP generator).
  6. Chemotransformation of E.coli TOP10 with ligation products.
  7. Plating transformants on LB+Amp30+X-gal+IPTG.
Preparation of constructs: OmpA_alpha and OmpA_omega

Michał K.

Clean-up of OmpA_alpha and OmpA_omega digest products.

Cloning alpha-A and omega-A fusions on pKS

Michał L., Ewa, Marcin

  1. Gel electophoresis of PCL products (Fig. 1).
  2. Gel-out of proper bands (alpha-A: 1100 bp and omega-A: 750 bp).
  3. Restriction digest of pKS vector and the PCL products with NotI and SacI (BamHI buffer) for 2 hours.
  4. Ligation 1 hour.
  5. Transformation of Top10 strain and screening on Amp100 plates.
Fig. 1.PCL product - Alpha-A fusion.


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