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Team:Warsaw/Calendar-Main/22 September 2008
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<h3>'Hunter and prey' system tests: Competition tests</h3> | <h3>'Hunter and prey' system tests: Competition tests</h3> | ||
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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> | <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> | ||
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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