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Team:Warsaw/Calendar-Main/22 September 2008
From 2008.igem.org
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[photo of the gel is to be placed here] | [photo of the gel is to be placed here] | ||
| - | <p>Conclusion: cells with | + | <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> | ||
| - | <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 | ||
Revision as of 18:24, 27 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 testsWeronika
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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