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.