• Construction of stability epitopes

  The main idea is to use the primers 57-58 and 59 as matrices in order to synthesize and amplify the stability epitope. The PCR uses the iGEM PCR protocol with Q5 polymerase. The temperature of annealing is 55°C, the elongation time is 5", the number of cycle is 25.

  Mix:

  • 5µL OiGEM-57 or 58 or 59
  • 2,5µL OiGEM-60
  • 2,5µL OiGEM-44
  • 25µL Q5 2x Mix
  • 15µL H2O
  

• Clean-up of these PCR and elution with 30µL H2O at 70°C.

• PCR cheA-kan-cheA using the iGEM PCR protocol with Q5 polymerase. The temperature of annealing is 60°C, the elongation time is 1’30”.

  Mix:

  • 1µL pKD4 vector
  • 2,5µL primer 3
  • 2,5µL primer 4
  • 2,5µL dNTP
  • 10µL Q5 buffer
  • qsp 50µL H2O
  

• Construction of stability epitopes

  Test 5 clones of each transformation (with pSB1C3- K10512-06 or 07 or 08) by PCR. The temperature of annealing is 55°C, the elongation time is 1'

  Mix:

  • 2µL OiGEM-60
  • 2µL OiGEM-44
  • 4µL dNTP
  • 1µL DMSO
  • 2µL buffer
  • 0,5µL Taq polymérase
  • 8,5µL H2O
  

  All clones are good except the K10512-08-2.

  The pSB1C3-K10512-06-1, pSB1C3-K10512-07-1 and pSB1C3-K10512-08-1 are sent to sequencing. They are OK.

• The bacteria are sensitive to kanamicyn.

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.

• CurR-box characterization

  • Digestion ES of cusR box BBa_K1349002 (x1 or x2), digestion XP of RBS-GFP (200(2)) and digestion EP of piGEM-01.13 (pSB1C3-BBa_J04450).

    500ng DNA
    1µL EcoRI or XbaI
    1µL SpeI or PstI
    5µL NEB Buffer 2.1
    qsp 50µL of water

    Incubation 60' at 37°C.

  • Then inactivation of the enzymes for 20’ at 80°C.

  • Ligation of the cusR box BBa_K1349002 (x1 or x2) with RBS-GFP (200(2)) in piGEM-01.13.

    2µL piGEM-01.13
    4µL BBa_K1349002 (x1 or x2)
    4µL 200(2)
    1µL T4 ligase 10U/µL
    2µL T4 ligase buffer
    7µL H2O

  • Digestion ES of cusR box BBa_K1349002 (x2), digestion XP of cusR box BBa_K1349002 (x1) and digestion EP of piGEM-01.13 (pSB1C3-BBa_J04450).

    500ng DNA
    1µL EcoRI or XbaI
    1µL SpeI or PstI
    5µL NEB Buffer 2.1
    qsp 50µL of water

    Incubation 60' at 37°C.

  • Then inactivation of the enzymes for 20’ at 80°C.

  • Ligation of the cusR box BBa_K1349002 (x2) with cusR box BBa_K1349002 (x1) in piGEM-01.13.

    2µL piGEM-01.13
    4µL BBa_K1349002 (x2)
    4µL BBa_K1349002 (x1)
    1µL T4 ligase 10U/µL
    2µL T4 ligase buffer
    7µL H2O

  • Transformation of 100µL of E.coli TG1 competent cells with 10µL of ligation. The selective medium is LB-Cm 30µg/mL.

• Each preculture was back-diluted 100x in modified M9 medium (see protocol section), cultivated at 37°C with continuous shaking until they reached an OD600nm=0,4 to 0,6. Then, a 5 mL aliquot of each culture was treated with a sub-lethal concentration of AgNO3 (5 μM, see reference Gudipaty SA et al., 2012) while a second untreated aliquot of 5 mL was used as a control. After Ag treatment, all the cultures were incubated in the dark at 37°C with continuous shaking. Then, cell growth and GFP fluorescence were determined over time by reading the OD at 600 nm and 530 nm, respectively. To do so, 150 μL aliquots were transferred into wells of a black 96-well plate (Greiner), and the absorbance at 600 nm and fluorescence (excitation, 485 nm; emission, 530 nm) were measured with a Tecan Infinite M200 microplate reader.

• Growth was determined overtime by following the culture OD at 600 nm on a TECAN instrument (Result section, Figure A).

• GFP synthesis was quantified by measuring the OD at 530 nm on a TECAN instrument. Then, fluorescence was normalized by dividing the results with the OD at 600 nm (Result section, Figure B).

See the result section for details on our interpretation of the data.