Team:Warsaw/Calendar-Main/29 July 2008

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Purification of proteins: Z-alpha and Z-omega

Piotr

pET15b+Z_alpha and pET15b+Z_omega in Rosetta strain inoculated in liquid LB with chloramphenicol and ampicillin.

Cloning of omega_ΔA DNA fragment to pACYC177+OmpA_alpha

Michał K.

Isolation of pKS+omega_ΔA from culture inoculated on previous day.
Digest of pKS+omega_ΔA with SacI and NotI (BamHI buffer).
Digest (SacI and NotI) and dephosphorylation (CIAP) of pACYC177+OmpA_alpha.
Gel electrophoresis and gel-out of 4300 bp (pACYC177+OmpA_alpha) and 550 bp (omega_ΔA) bands (Fig. 1.).
Ligation of DNA fragments from 2. and 3. (1 hr).
Transformation of E. coli TOP10 strain with ligation.
Transformants plating on LB + kanamycin.

Fig. 1. SacI/NotI digests of isolated plasmids 1. Marker 2. digested pKS_omega_ΔA 3. digested pACYC_Omp_alpha

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-<p>1. Separate transformant colonies (transformation from previous day) inoculated to liquid LB with kanamycin. <br>
-. Inoculation of pACYC177+OmpA_alpha and pACYC177+OmpA_omega - liquid LB with kanamycin.</p>
-Piotr:
+<h3>Purification of proteins: Z-alpha and Z-omega</h3><h4>Piotr</h4>
-Checking if OmpA_omega_A_alpha gives ampicillin resistance.
+<p><A href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BZ-alpha>pET15b+Z_alpha</A> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2Bhis%2BZ%2Bomega>pET15b+Z_omega</a> in <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#rosetta">Rosetta</a> strain inoculated in liquid LB with chloramphenicol and ampicillin.
-Inoculation OmpA_omega_A_alpha from various IPTG concentrations (0, 0.1, 0.25, 0.5, 0.75, 1) into the same IPTG concentrations, but with various ampicillin concentrations(25, 50, 75, 100) in relation: 1:50.
+</p>
+<h3>Cloning of omega_&Delta;A DNA fragment to <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha>pACYC177+OmpA_alpha</a></h3><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/pKSII%2Bomega-deltaA>pKS+omega_&Delta;A</a> from culture inoculated on previous day.</li>
+<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>Digest</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pKSII%2Bomega-deltaA>pKS+omega_&Delta;A</a>  with SacI and NotI (BamHI buffer).</li>
+<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>Digest</a> (SacI and NotI) and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">dephosphorylation</a> (CIAP) of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha>pACYC177+OmpA_alpha</a>. </li>
+<li>Gel electrophoresis and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of 4300 bp (<a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha>pACYC177+OmpA_alpha</a>) and 550 bp (omega_&Delta;A) bands (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/29_July_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 DNA fragments from 2. and 3. (1 hr).</li>
+<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">Transformation</a> of E. coli <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> strain with ligation.</li>
+<li>Transformants plating on LB + kanamycin.</li></ol>
+</p>
+<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/6/6a/Po_go.jpg" width=200/></a> <var><b>Fig. 1. </b> SacI/NotI digests of isolated plasmids<br>
+. Marker<br>
+. digested pKS_omega_&Delta;A<br>
+. digested pACYC_Omp_alpha<br></var>
-Measurement of bacterial culture growth (OD) in the evening:
-<table id="result" align="center">
-<tr><th rowspan="2">ampicillin concentration (mikrogramy na mililitr):</th><th colspan="6">IPTG concentration (milimole na mililitr):</td></tr>
-<tr><th>0</th><th>0,1</th><th>0,25</th><th>0,5</th><th>0,75</th><th>1</th></tr>
-<tr><th>25</th><td class="live">1,558</td><td class="live">1,469</td><td class="live">1,587</td><td class="live">1,49</td><td class="live">1,566</td><td class="live">1,311</td></tr>
-<tr><th>50</th><td class="live">1,425</td><td class="live">1,435</td><td class="live">1,524</td><td class="live">1,055</td><td class="live">0,920</td><td class="live">0,935</td></tr>
-<tr><th>75</th><td class="live">1.09</td><td class="live">0,989</td><td class="live">1.447</td><td class="live">0.971</td><td class="live">0.951</td><td class="live">0.992</td></tr>
-<tr><th>100</th><td class="live">0.09</td><td class="live">0,685</td><td class="live">1.378</td><td class="live">1.078</td><td class="live">0.977</td><td class="live">0.992</td></tr>
-</table>
-Inoculation of OmpA_omega_A_alpha into various IPTG concentrations: 0, 0.1, 0.25, 0.5, 0.75 (repeat is needed!!!)
-Checking OmpA_omega_A_alpha and OmpA_A_alpha expression.
-<ol>
-<li>Spinnign</li>
-<li>Suspending</li>
-<li>Adding of lysis buffer</li>
-<li>Boiling</li>
-<li>Putting into poliacrylamide gel</li>
-<li>Transfer onto nitrocellulose</li>
-<li>Blocking</li>
-<li>Anti-A antibody binding</li>
-<li>Washing</li>
-<li>Anti-rabbit antibody binding</li>
-<li>Developing with BCIP and NBT</li>
-</ol>
-(Zdjęcie żelu)
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