Team:Warsaw/Calendar-Main/22 September 2008

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<h3>MutD<sub>5</sub> testing</h3>
<h3>MutD<sub>5</sub> testing</h3>
<h4>Piotr</h4>
<h4>Piotr</h4>
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<p>Inoculation MutD<sub>5</sub> carrying: OmpA_Z_alpha, OmpA_Z_omega, Omp_A_omega and OmpA_omega_A + induction using 0.25mM IPTG.</p>
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<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_&Delta;A</a> + induction using 0.25mM IPTG.</p>
<h3>'Hunter and prey' system tests: Competition tests</h3>
<h3>'Hunter and prey' system tests: Competition tests</h3>
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<h4>Weronika</h4>
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<h4>Emilia</h4>
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<p><ol><li>Plasmid isolation from 19 September cultures.</li><li>Digest with SacI and BamHI.</li><li>Electrophoresis.</li></ol></p>
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<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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[photo of the gel is to be placed here]
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/5/51/Konkurencja2.jpg"/></a>
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<p>Conclusion: cells with interracting protein survive competition!</p>
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<var><b>Fig. 1.</b> Results of the second competition test.<br>
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1 - Insert from isolated plasmid refers to OmpA_A_Omega <br>(Z_Alpha protein added to the medium),<br>
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2 - Insert from isolated plasmid refers to OmpA_A_Alpha <br>(Z_Omega protein added to the medium),<br>
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3 - Insert from isolated plasmid refers to OmpA_Z_Omega <br>(A_Alpha protein added to the medium),<br>
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4 - DNA ladder.</var>
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<p>Conclusion: cells with interacting protein survive competition!</p>
<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>
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<p>Mutagenesis were performed on 3 vectors:  <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-A-omega>pACYClac+ompA-A-omega</a>, <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-A-alpha>pACYClac+ompa-A-alpha</a> and pACYClac+ ompa-alpha-A.</p>
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<p>Mutagenesis was performed on 3 vectors:  <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BOmpA-deltaA-omega>pACYClac+ompA-&Delta;A-omega</a>, <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-deltaA-alpha>pACYClac+ompa-&Delta;A-alpha</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-deltaA>pACYClac+ ompa-omega-&Delta;A</a>.</p>
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<p>Each mutagenesis was performed using each primer at final concentration 0.1 μM, dNTPs at final concentration 0.25 μM and Walk (Pwo) polymerase (shipped by <a href=http://aabiot.com/>A&A Biotechnology</a>), with 100 ng of DNA template. </p>
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<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&deg;C 5 min
<pre style="text-align: left">94&deg;C 5 min
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</pre></p>
</pre></p>
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<h3>Preparation of BioBricks</h3>
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<h3>Preparation of <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103003>&Delta;A (BBa_K103003)</a></h3>
<h4>Michał K.</h4>
<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>) - RFP(??????)+deltaA plasmid into liquid LB + ampicillin. </p>  
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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>&Delta;A</a>plasmid into liquid LB + ampicillin. </p>  
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MutD5 testing

Piotr

Inoculation 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 tests

Emilia

  1. Plasmid isolation from 19 September cultures.
  2. Digest with SacI and BamHI.
  3. Electrophoresis (Fig. 1.) .

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 A

Paweł

Mutagenesis 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.


PCR program (15 cycles): 

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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