Team:Warsaw/Calendar-Main/22 September 2008

MutD5 testing Piotr Inoculation of MutD5 carrying: pACYC177+OmpA_Z_alpha, pACYC177+OmpA_Z_omega, pACYC177+Omp_A_omega and pACYC177+OmpA_omega_&Delta;A + induction using 0.25mM IPTG.

'Hunter and prey' system tests: Competition tests Emilia Plasmid isolation from 19 September cultures.Digest with SacI and BamHI.</li>Electrophoresis (Fig. 1.</a>) .</li></ol>

<img src="http://2008.igem.org/wiki/images/5/51/Konkurencja2.jpg"/></a> 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 A Paweł Mutagenesis of protein A was performed using 2 pairs of primers: ADelL+KpnI</a> and ADelP</a> (deletion of aminoacids involved in interaction with protein Z) and AMutL+KpnI</a> and AMutP</a> (changing of few aminoacids involved in interaction with protein Z). Mutagenesis was performed on 3 vectors: pACYClac+ompA-&Delta;A-omega</a>, pACYClac+ompa-&Delta;A-alpha</a> and pACYClac+ ompa-omega-&Delta;A</a>. 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</a>), with 100 ng of DNA template. PCR program (15 cycles): <pre style="text-align: left">94&deg;C 5 min

94&deg;C 30 s 55&deg;C 30 s 72&deg;C 10 min

72&deg;C 8 min

4&deg;C hold

Preparation of &Delta;A (BBa_K103003)</a> Michał K.

Inoculation of only one colony which grown from transformation (19 September</a>) - pSB1A3</a>+&Delta;A</a>plasmid into liquid LB + ampicillin.