Team:Warsaw/Calendar-Main/22 September 2008

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MutD5 testing

Piotr

Inoculation 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 tests

Weronika

  1. Plasmid isolation from 19 September cultures.
  2. Digest with SacI and BamHI.
  3. Electrophoresis (fig. 1.) .

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 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.


PCR program (15 cycles):

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.