Team:INSA Toulouse/contenu/lab practice/notebook/calendar/MG1655 – 2013.igem.org

  • 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.

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    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.

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    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.

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    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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