Team:INSA Toulouse/contenu/lab practice/notebook/calendar/MG1655

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MG1655 del(EnvZ) Strain

August 2013

  • Week 9 (5-11 August)
    In order to use this strain for light sensors characterization, we need to delete the envZ gene. For this we did a transduction of the strain with phages previously mixed with the del(EnvZ) strain from the Keio collection.
    The strains from this collection have individual kan cassette replacements of non-essential genes flanked by FRT sites allowing excision using the FLP recombinase.
    6/09
    overnight culture of the Keio Del(EnvZ) Strain in LB and CaCl2

    7/09
    dilution of the overnight culture of del(envZ) strain until DO=1
    a mix of bacterias with P1 phages is spread on soft LB agar for the night at 30°
    overnight culture of the MG1655 strain

    8/09
    pick up of phage with bacterias
    dilution of the overnight culture of MG1655 in LB and CaCl2 until DO=1
    mix of bacterias (MG1655) with phages
    incubation a night at 37°c

    9/09
    verification of lysis plaque


  • Week 10 (12-18 August)
    Then we needed to remove the Kn cassette with the FLIP recombinase, in order to integrate with a selection factor Kn.

    12/09
    Transformation with the pFLP plasmid.
    One night on LB agar plate at 30°C

    13/09
    Pick 4 clones and cultivate in liquid 2hours at 30°C then 3 hours at 42°C.
    Dilution and spread on LB Agar.

    14/09
    Pick isolated clones on LB Agar, LB Cm, LB Ap and LB Kn.
    Let one night at 42°C.
    Right clones are Cm S, Ap S and Kn S.

    15/08
    Clones are ok.
    Waiting for the primers to verify with PCR.
  • Week 11 (19-25 August)
    19/08
    PCR verification: ok ! The del(envZ) deFRT Kn is constructed!! Thanks to the wonderful help of Caroline Schiavon


    20/08
    Then in order to integrate modules in the genome with integration plasmids, the strain needs to be Streptomycine resistant for selection.
    The strain has the Streptomycine resistance gene but with a mutation. With several spread on LB agar Strp, the strain can recover the resistance.
    Spread on LB Str 50ng during a night at 37°c.

    21/08
    Several clones have grown but the right Strepto concentration was 200ng/µL.
    Spread on LB Str 200ng during a night at 37°C

    22/08
    One clone on the plate
    Subcloned the clone on spread on LB Str 200ng over the night at 37°C

    23/08
    Few other clones have grown
    Subcloned of these clones and spread on LB Str 200ng at 37°C

    25/08
    Competent cells of the new strain: MG1655 del(envZ) deFRT Kn StrpR.
    Transformation with the plasmid pMS58
    Spread on LB Cm over the night at 30°C
  • Week 12 (26-1 September)
    26/08
    streak 4 clones of the previous transformation on LB Cm at 42°C one night

    27/08
    Streak 4 clones of the first integration on LB Cm at 42°C one night

    28/08
    Streak 4 clones of the second integration on LB Strp 42°C one night

    29/08
    Sub-cloned few clones on LB Cm, LB Str and LB Kn at 42°C one night
    Right clone are StR CmS et KnR

    30/08
    Our clones were StrR, but unfortunately they were CmR and KnS…
    Restart of the integration from the beginning:
    Making competent cells
    In parallele, spread of the MG1655 del(envZ) deFRT Kn StrpR strain on LB Cm, LB Str and LB Kn at 42°C

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