Team:Aix-Marseille/Notebook relA

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Notebook: RelA



Week 13 : 09/22/2014 ➡ 09/28/2014

Attribution: Emmanuelle Bouveret
Objective: Test the functionality of our RelA part.

The pSB1K3-relA plasmid constructed by the iGEM-AMU team is expected to be able to rescue the phenotype of a MG1655∆relA mutant grown on SMG plates, a medium known to induce a strong amino acid depletion in the cells (Rudd et al.,1985) that is toxic to a ∆relA mutant.



  • Day 1:

    Isolated clones were restreaked on SMG plates supplemented with 50 uM Kanamycin and 0.5mM IPTG. The plates were incubated at 37°C during 48 hrs.

  • Day 2:

    Electrocompetent MG1655∆relA (EB421 strain, Wahl et al. 2011) cells were electroporated with either plasmid pSB1K3-RFP as a control or pSB1K3-relA.

  • Day 4:

    As expected, we observed that the relA construct was able to complement the MG1655∆relA strain, as seen on the following figure.

  • SMG plate composition:

    M9 salts 1X
    MgSO4 1mM
    CaCl2 0.1mM
    VitB1 0.5µg/ml
    Glucose 0.2%
    Serine 1mM
    Methionine 1mM
    Glycine 1mM
    Bactoagar 15g/L

    References:

    • Rudd KE, Bochner BR, Cashel M, Roth JR. Mutations in the spoT gene of Salmonella typhimurium: effects on his operon expression. J Bacteriol. ;163(2):534-42, 1985.
    • Wahl A, My L, Dumoulin R, Sturgis JN, Bouveret E.Antagonistic regulation of dgkA and plsB genes of phospholipid synthesis by multiple stress responses in Escherichia coli. Mol Microbiol. 80(5):1260-75, 2011.