Team:Warsaw/Calendar-Main/17 September 2008

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'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.

PCR on pACYC177+OmpA_omega_ΔA_alpha plasmid using AL_BNXNE and APSacSpe primers (elongation length 45s) to obtain ΔA fragment.
PCR on pACYC177+OmpA_omega_ΔA_alpha plasmid using LinLSXNE and AlfaPSpe primers (elongation length 60s) to obtain link_alpha fragment.
PCR on pACYC177 + OmpA_omega plasmid using LinLSXNE and OmegPSpe primers (elongation length 60s) to obtain link_omega fragment.
PCR on pACYC177+OmpA_omega_ΔA_alpha plasmid using OmpLNXNE and LinP_BS primers (elongation length 90s) to obtain OmpA_omega fragment.

Each reaction was carried out with 25 cycles and in temperature gradient from 55 to 75 °C (four reactions).

Gel electrophoresis of PCR products (Fig. 2.).

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