Team:Warsaw/Calendar-Main/17 September 2008

'Hunter and prey' system tests: Competition tests Piotr Plasmid isolation from previous day's cultures.

Digest with SacI and BamHI.Electrophoresis (Fig. 1).

 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 testing Emilia Inoculation of MutD5 into medium with tetracycline.

Optimisation of primers for preparation of parts

Michał K. <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on <a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>pACYC177+OmpA_omega_&Delta;A_alpha</a> plasmid using

<a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#AL_BNXNE">AL_BNXNE</a> and <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#APSacSpe">APSacSpe</a>

primers (elongation length 45s) to obtain &Delta;A fragment. </li>  <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on <a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>pACYC177+OmpA_omega_&Delta;A_alpha</a> plasmid using

<a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#LinLSXNE">LinLSXNE</a> and <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#AlfaPSpe">AlfaPSpe</a> primers (elongation length 60s) to obtain link_alpha fragment. </li> <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on <a href="http://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega">pACYC177 + OmpA_omega</a> plasmid using <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#LinLSXNE">LinLSXNE</a> and <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#OmegPSpe">OmegPSpe</a> primers (elongation length 60s) to obtain link_omega fragment. </li>

<a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on <a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>pACYC177+OmpA_omega_&Delta;A_alpha</a> plasmid using <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#OmpLNXNE">OmpLNXNE</a> and <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#LinP_BS">LinP_BS</a> primers (elongation length 90s) to obtain OmpA_omega fragment. </li> Each reaction was carried out with 25 cycles and in temperature gradient from 55 to 75 &deg;C (four reactions).  Gel electrophoresis of PCR products (<a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/17_September_2008#fig2">Fig. 2.</a>).</li> </ol>

<img src="http://2008.igem.org/wiki/images/9/9e/Grad_17_09.jpg" width=300/></a> Fig. 2. Gradient PCR (temperatures:55-75 &deg;C) 1. Marker 2. 55 &deg;C deltaA 3. 60 &deg;C deltaA 4. 65 &deg;C deltaA 5. 70 &deg;C deltaA 6. 55 &deg;C link_omega 7. 60 &deg;C link_omega 8. 65 &deg;C link_omega 9. 70 &deg;C link_omega 10. 55 &deg;C link_alpha 11. 60 &deg;C link_alpha 12. 65 &deg;C link_alpha 13. 70 &deg;C link_alpha 14. 55 &deg;C OmpA_omega 15. 60 &deg;C OmpA_omega 16. 65 &deg;C OmpA_omega 17. 70 &deg;C OmpA_omega