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Team:Warsaw/Calendar-Main/17 September 2008
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| + | <h3>'Hunter and prey' system tests: Competition tests</h3> | ||
| + | <h4>Piotr</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 previous day's 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/17_September_2008#fig1">Fig. 1</a>).</li></ol></p> | ||
| - | + | <a name="fig1"><img src="https://static.igem.org/mediawiki/2008/9/98/Konkurencja1.jpg" width=180/></a> | |
| - | + | <var><b>Fig. 1.</b> Result of competition test:<br> | |
| - | + | 1 and 5 - DNA ladder,<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_A_Omega <br>(Z_Alpha protein added to the medium),<br> | ||
| + | 4 - Insert from isolated plasmid refers to OmpA_Z_Omega <br>(A_Alpha protein added to the medium).</var> | ||
| - | + | <p>Conclusion: cells with interacting protein survive competition!</p> | |
| - | + | <h3>MutD5 testing</h3> | |
| + | <h4>Emilia</h4> | ||
| + | <p>Inoculation of MutD5 into medium with tetracycline.</p> | ||
| + | <h3>Optimisation of primers for preparation of parts</h3> | ||
| + | <h4>Michał K.</h4> | ||
| + | <ol><li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>pACYC177+OmpA_omega_ΔA_alpha</a> plasmid using | ||
| - | + | <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL_BNXNE">AL_BNXNE</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#APSacSpe">APSacSpe</a> | |
| - | + | primers (elongation length 45s) to obtain ΔA fragment. </li> | |
| + | <li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>pACYC177+OmpA_omega_ΔA_alpha</a> plasmid using | ||
| + | <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#LinLSXNE">LinLSXNE</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlfaPSpe">AlfaPSpe</a> | ||
| + | primers (elongation length 60s) to obtain link_alpha fragment. </li> | ||
| + | <li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on <a href="https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega">pACYC177 + OmpA_omega</a> plasmid using | ||
| + | <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#LinLSXNE">LinLSXNE</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmegPSpe">OmegPSpe</a> | ||
| + | primers (elongation length 60s) to obtain link_omega fragment. </li> | ||
| - | < | + | <li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>pACYC177+OmpA_omega_ΔA_alpha</a> plasmid using |
| + | <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmpLNXNE">OmpLNXNE</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#LinP_BS">LinP_BS</a> | ||
| + | primers (elongation length 90s) to obtain OmpA_omega fragment. </li> | ||
| + | <p>Each reaction was carried out with 25 cycles and in temperature gradient from 55 to 75 °C (four reactions).</p> | ||
| + | <li> Gel electrophoresis of PCR products (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/17_September_2008#fig2">Fig. 2.</a>).</li> | ||
| + | </ol> | ||
| - | < | + | <a name="fig2"><img src="https://static.igem.org/mediawiki/2008/9/9e/Grad_17_09.jpg" width=300/></a><var><b>Fig. 2.</b> Gradient PCR (temperatures:55-75 °C)<br> |
| - | + | 1. Marker<br> | |
| + | 2. 55 °C deltaA<br> | ||
| + | 3. 60 °C deltaA<br> | ||
| + | 4. 65 °C deltaA<br> | ||
| + | 5. 70 °C deltaA<br> | ||
| + | 6. 55 °C link_omega<br> | ||
| + | 7. 60 °C link_omega<br> | ||
| + | 8. 65 °C link_omega<br> | ||
| + | 9. 70 °C link_omega<br> | ||
| + | 10. 55 °C link_alpha<br> | ||
| + | 11. 60 °C link_alpha<br> | ||
| + | 12. 65 °C link_alpha<br> | ||
| + | 13. 70 °C link_alpha<br> | ||
| + | 14. 55 °C OmpA_omega<br> | ||
| + | 15. 60 °C OmpA_omega<br> | ||
| + | 16. 65 °C OmpA_omega<br> | ||
| + | 17. 70 °C OmpA_omega<br></var> | ||
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'Hunter and prey' system tests: Competition testsPiotr
Fig. 1. Result of competition test:1 and 5 - DNA ladder, 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_A_Omega (Z_Alpha protein added to the medium), 4 - Insert from isolated plasmid refers to OmpA_Z_Omega (A_Alpha protein added to the medium). Conclusion: cells with interacting protein survive competition! MutD5 testingEmiliaInoculation of MutD5 into medium with tetracycline. Optimisation of primers for preparation of partsMichał K.
Each reaction was carried out with 25 cycles and in temperature gradient from 55 to 75 °C (four reactions).  Fig. 2. Gradient PCR (temperatures:55-75 °C)1. Marker 2. 55 °C deltaA 3. 60 °C deltaA 4. 65 °C deltaA 5. 70 °C deltaA 6. 55 °C link_omega 7. 60 °C link_omega 8. 65 °C link_omega 9. 70 °C link_omega 10. 55 °C link_alpha 11. 60 °C link_alpha 12. 65 °C link_alpha 13. 70 °C link_alpha 14. 55 °C OmpA_omega 15. 60 °C OmpA_omega 16. 65 °C OmpA_omega 17. 70 °C OmpA_omega
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