Team:Warsaw/Calendar-Main/21 August 2008

Tests for ampicillin resistance given by protein added to medium Piotr Results of growth from previous day

 Isolation of plasmids from above cultures.   Control digest of isolated plasmids with BamHI and SacI.  Gel electrophoresis - all constructs apart from pACYC177+OmpA_alpha were successfully recovered.  Conclusions:  Almost none of tested constructs give resistance to ampicillin without prey protein added to culture medium.</li> Interaction between hunter and prey is not necessary to make cells ampicillin resistant (OmpA_alpha and OmpA_omega grow with prey).</li> <a href=http://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>

Cloning of protein A DNA to GeneArt plasmid Antoni   Colony <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on colonies from plates with transformations <A href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pGeneart-A>pGeneart+A</a>. Primers used:

<a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a> and <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#AL+SacI">AL+SacI</a>. </li> Gel electrophoresis.</li>  Confirmed transformant colonies inoculated to liquid LB with ampicillin.</li>

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Cloning of alpha to <a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-A-omega>pACYC177+OmpA_A_omega</a> and <a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-Z-omega>pACYC177+OmpA_Z_omega</a> Michał K.    <a href="http://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>  Control <a href="http://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digest</a> of isolated plasmids with FastBamHI and FastAcc65I (Fast Digest buffer).</li>  Gel electrophoresis - no proper clones found (<a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/21_August_2008#fig1">Fig. 1.</a>).</li> </ol>

<img src="http://2008.igem.org/wiki/images/f/f3/Traw_04_09_2008.jpg" width=300/></a> 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