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Team:Warsaw/Calendar-Main/22 September 2008
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| + | <h3>MutD<sub>5</sub> testing</h3> | ||
| + | <h4>Piotr</h4> | ||
| + | <p>Inoculation of MutD<sub>5</sub> carrying: <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-Z-alpha>pACYC177+OmpA_Z_alpha</a>, <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-Z-omega>pACYC177+OmpA_Z_omega</a>, <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-A-omega>pACYC177+Omp_A_omega</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-deltaA>pACYC177+OmpA_omega_ΔA</a> + induction using 0.25mM IPTG.</p> | ||
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| + | <h3>'Hunter and prey' system tests: Competition tests</h3> | ||
| + | <h4>Emilia</h4> | ||
| + | <p><ol><li>Plasmid <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation>isolation</a> from 19 September cultures.</li><li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>Digest</a> with SacI and BamHI.</li><li>Electrophoresis (<a href="https://2008.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/22_September_2008#fig1">Fig. 1.</a>) .</li></ol></p> | ||
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| + | <a name="fig1"><img src="https://static.igem.org/mediawiki/2008/5/51/Konkurencja2.jpg"/></a> | ||
| + | <var><b>Fig. 1.</b> Results of the second competition test.<br> | ||
| + | 1 - Insert from isolated plasmid refers to OmpA_A_Omega <br>(Z_Alpha protein added to the medium),<br> | ||
| + | 2 - Insert from isolated plasmid refers to OmpA_A_Alpha <br>(Z_Omega protein added to the medium),<br> | ||
| + | 3 - Insert from isolated plasmid refers to OmpA_Z_Omega <br>(A_Alpha protein added to the medium),<br> | ||
| + | 4 - DNA ladder.</var> | ||
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| + | <p>Conclusion: cells with interacting protein survive competition!</p> | ||
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<h3>Mutagenesis of protein A</h3><h4>Paweł</h4> | <h3>Mutagenesis of protein A</h3><h4>Paweł</h4> | ||
<p>Mutagenesis of protein A was performed using 2 pairs of primers: <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/primers#ADelL+KpnI>ADelL+KpnI</a> and <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/primers#ADelP>ADelP</a> (deletion of aminoacids involved in interaction with protein Z) and <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/primers#AmutL+KpnI>AMutL+KpnI</a> and <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/primers#AmutP>AMutP</a> (changing of few aminoacids involved in interaction with protein Z).</p> | <p>Mutagenesis of protein A was performed using 2 pairs of primers: <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/primers#ADelL+KpnI>ADelL+KpnI</a> and <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/primers#ADelP>ADelP</a> (deletion of aminoacids involved in interaction with protein Z) and <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/primers#AmutL+KpnI>AMutL+KpnI</a> and <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/primers#AmutP>AMutP</a> (changing of few aminoacids involved in interaction with protein Z).</p> | ||
| - | <p>Mutagenesis | + | <p>Mutagenesis was performed on 3 vectors: <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BOmpA-deltaA-omega>pACYClac+ompA-ΔA-omega</a>, <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-deltaA-alpha>pACYClac+ompa-ΔA-alpha</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-deltaA>pACYClac+ ompa-omega-ΔA</a>.</p> |
| - | <p>Each mutagenesis was performed using each primer at final concentration 0.1 μM, dNTPs at final concentration 0.25 μM and Walk ( | + | <p>Each mutagenesis was performed using each primer at final concentration 0.1 μM, dNTPs at final concentration 0.25 μM and Walk (Pfu) polymerase (shipped by <a href=http://aabiot.com/>A&A Biotechnology</a>), with 100 ng of DNA template. </p> |
<p><br>PCR program (15 cycles): | <p><br>PCR program (15 cycles): | ||
<pre style="text-align: left">94°C 5 min | <pre style="text-align: left">94°C 5 min | ||
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4°C hold | 4°C hold | ||
</pre></p> | </pre></p> | ||
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| + | <h3>Preparation of <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103003>ΔA (BBa_K103003)</a></h3> | ||
| + | <h4>Michał K.</h4> | ||
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| + | <p> Inoculation of only one colony which grown from transformation (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/19_September_2008">19 September</a>) - <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a>+<a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103003>ΔA</a>plasmid into liquid LB + ampicillin. </p> | ||
</html> | </html> | ||
Latest revision as of 21:12, 29 October 2008
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MutD5 testingPiotrInoculation of MutD5 carrying: pACYC177+OmpA_Z_alpha, pACYC177+OmpA_Z_omega, pACYC177+Omp_A_omega and pACYC177+OmpA_omega_ΔA + induction using 0.25mM IPTG. 'Hunter and prey' system tests: Competition testsEmilia
Fig. 1. Results of the second competition test.1 - Insert from isolated plasmid refers to OmpA_A_Omega (Z_Alpha protein added to the medium), 2 - Insert from isolated plasmid refers to OmpA_A_Alpha (Z_Omega protein added to the medium), 3 - Insert from isolated plasmid refers to OmpA_Z_Omega (A_Alpha protein added to the medium), 4 - DNA ladder. Conclusion: cells with interacting protein survive competition! Mutagenesis of protein APawełMutagenesis of protein A was performed using 2 pairs of primers: ADelL+KpnI and ADelP (deletion of aminoacids involved in interaction with protein Z) and AMutL+KpnI and AMutP (changing of few aminoacids involved in interaction with protein Z). Mutagenesis was performed on 3 vectors: pACYClac+ompA-ΔA-omega, pACYClac+ompa-ΔA-alpha and pACYClac+ ompa-omega-ΔA. Each mutagenesis was performed using each primer at final concentration 0.1 μM, dNTPs at final concentration 0.25 μM and Walk (Pfu) polymerase (shipped by A&A Biotechnology), with 100 ng of DNA template.
94°C 5 min 94°C 30 s 55°C 30 s 72°C 10 min 72°C 8 min 4°C hold Preparation of ΔA (BBa_K103003)Michał K.Inoculation of only one colony which grown from transformation (19 September) - pSB1A3+ΔAplasmid into liquid LB + ampicillin.
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