Team:Warsaw/Calendar-Main/22 September 2008

From 2008.igem.org

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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>
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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>
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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 were 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>
<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>
<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>
<p><br>PCR program (15 cycles):
<p><br>PCR program (15 cycles):
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<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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Revision as of 23:19, 25 October 2008

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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

Weronika

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

[photo of the gel is to be placed here]

Conclusion: cells with interracting 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 were 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 (Pwo) 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 BioBricks

Michał K.

Inoculation of only one colony which grown from transformation (19 September) - pSB1A3+ΔAplasmid into liquid LB + ampicillin.


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