Team:Warsaw/Calendar-Main/21 August 2008

From 2008.igem.org

Revision as of 17:56, 24 October 2008 by Emilia (Talk | contribs)

Gallery Bricks Notebook Team Project Home


Previous day
return to main notebook page
Previous entry
next notebook entry

 


Tests for ampicillin resistance given by protein added to medium

Piotr

Results of growth from previous day

OmpA variant ("hunter")"Prey" variantGrowth with preyGrowth without prey
OmpA-alphaHis+Z+omega++
OmpA-alphaHis+Z+alpha++
OmpA-A-alphaHis+Z+omega+-
Ompa-A-omegaHis+Z+alpha+-
OmpA-omegaHis+Z+alpha+-
Ompa-Z-alphaHis+Z+omega+-
OmpA_Adelta_alphaHis+Z+omega+-
OmpA_Adelta_omegaHis+Z+alpha+-
OmpA_omega_AdeltaHis+Z+alpha+-
OmpA-omega-A-alphano++


  1. Isolation of plasmids from above cultures.
  2. Control digest of isolated plasmids with BamHI and SacI.
  3. Gel electrophoresis - all constructs apart from ompa_alfa were successfully recovered


Conclusions:

  1. Almost none of tested constructs give resistance to ampicillin without prey protein added to culture medium.
  2. Interaction between hunter and prey is not necessary to makes cells ampicillin resistant(Omp_alpha and Omp_omega growth with prey)
  3. OmpA-alpha may be contaminated with antibiotic-resistant bacteria, alpha do not need interaction of A and Z to bind to omega - maybe we must give bacteria less prey

  4. Cloning of protein A DNA to GeneArt plasmid

    Antoni

    1. Colony PCR on colonies from plates with transformations pGeneart+A.
      Primers used: AP+NotI and AL+SacI.
    2. Gel electrophoresis.
    3. Confirmed transformant colonies inoculated to liquid LB with ampicillin.
    4. Inoculation to liquid LB with ampicillin: pET15b+OmpA-alpha.

    Cloning of alpha to pACYC177+OmpA_A_omega and pACYC177+OmpA_Z_omega

    Michał K.

    1. Isolation of plasmids from cultures inoculated on previous day.
    2. Control digest of isolated plasmids with FastBamHI and FastAcc65I (Fast Digest buffer).
    3. Gel electrophoresis - no proper clones found.