Team:Warsaw/Calendar-Main/13 June 2008

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Blue/white and rifampicin test #1

Piotr

Results of rifampicin test
StrainInducer / repressorNumber of colonies on rifampicinOD
Top10none32.75
Top10 pMPM-AID0.1% Arabinose632.03
Top10 pMPM-AIDnone32.4
Top10 pMPM-AID+T70.1% Arabinose1271.71
Top10 pMPM-AID+T7none72.27
Top10 pMPM-AIDT70.1% Arabinose202.53
Top10 pMPM-AIDT7none142.18

AID in transcription fusion works like AID, but induce slower growth than AID;
AID in translation fusion do not induce, or weakly induce rifampicin resistance mutation

Preparation of constructs: OmpA_alpha and OmpA_omega

Michał K.

  1. PCR on pB30D plasmid using OmpaL_N and OmpaP_link primers (elongation length 45s). Fig. 1 and Fig. 2.
  2. PCR on pUC19 plasmid using AlphaL_link and AlphaP_XB primers (elongation length 45s). Fig. 2 and Fig. 3.
  3. PCR on pUC19 plasmid using OmegaL_link and OmegaP_EPB primers (elongation length 30s). Fig. 3 and Fig. 4.
  4. Each reaction was carried out with 15 and 20 cycles and in temperature gradient from 55 to 75 degrees.




    Gradient PCR products: 1, 14 and 27-DNA ladder;
    2 to 8 - gradient PCR for Alpha carried out with 15 cycles;
    9 to 16 - gradient PCR for Alpha carried out with 20 cycles;
    17 to 23 - gradient PCR for Omega carried out with 15 cycles;
    24 to 31 - gradient PCR for Omega carried out with 20 cycles;
    32 to 38 - gradient PCR for OmpA carried out with 15 cycles;
    39 to 46 - gradient PCR for OmpA carried out with 20 cycles.
    Product od each variant of PCR is in 7 lanes: 1st is the lower border annealing temperature (55 degrees), 6th is the higher annealing temperature (75 degrees) and 7th is negative control without template DNA.