• CurR-box characterization : introduction of the CusR-box part (BBa_K1349002) into a pUC18 plasmid expressing the GFP from a pLAC promoter.

  The insertion of the BBa_K1349002 part downstream of the Plac promoter is expected to allow the binding of the CusR regulator in response to metal (Cu or Ag) availability in the medium. In our construct, we expect CusR binding to the CusR-boxes to physically blocking RNA polymerase, donc consequently to down regulate the expression of the GFP gene.

• CurR-box characterization

  • Digestion ES and XP of cusR box BBa_K1349002 and digestion EP of piGEM-02.28 (pSB1K3-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 digested ES with cusR box digested XP in piGEM-02.28 digested EP.

  2µL piGEM-02.28
  4µL BBa_K1349002 (ES)
  4µL BBa_K1349002 (XP)
  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-Km 25µg/mL.

• 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.

• CurR-box characterization

  • 2 clones of each ligation from the 06-10-14 are tested by digestion

    

    The clone 1 of each construction is used for the next steps.

  • Digestion EP of cusR box (cusBx1)-GFP, cusBx2-GFP and digestion EP of pUC18.

    1µg DNA
    1µL EcoRI (Promega)
    1µL PstI (Promega)
    5µL Buffer H of Promega
    5µL BSA
    qsp 50µL of water

    Incubation 60' at 37°C.

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

  • Ligation of the cusBx1-GFP or cusBx2-GFP with pUC18.

    1µL pUC18
    2µL cusBx1-GFP or cusBx2-GFP
    0,5µL T4 ligase 10U/µL
    2µL T4 ligase buffer
    14,5µL H2O

    Incubation ON at 16°C

  • Digestion ES of cusBx3, digestion XP of RBS-GFP (200(2)) and digestion EP of pUC18.

    1µg DNA
    1µL EcoRI or XbaI(Promega)
    1µL PstI or SpeI (Promega)
    5µL Buffer H of Promega
    5µL BSA
    qsp 50µL of water

    Incubation 60' at 37°C.

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

  • Ligation of cusBx3 with RBS-GFP (200(2)) in pUC18.

    1µL pUC18
    2µL cusBx3
    2µL RBS-GFP (200(2))
    0,5µL T4 ligase 10U/µL
    2µL T4 ligase buffer
    12,5µL H2O

    Incubation ON at 16°C

• Transformation of 100µL of E.coli TG1 competent cells with 10µL of ligation. The selective medium is LB-Ap 50µg/mL-IPTG 100µg/mL-X-galactose 75µg/mL.

• A green colony for each ligation from the 08-10-14 is streaked out on LB-Ap 50µg/mL-IPTG 100µg/mL-X-galactose 75µg/mL.

• Preculture of the different strains of interest in LB medium + Ap100 at 37°C O/N

  • TG1 pUC18-GFP: expresses GFP from a Plac leaky promoter on the pUC18 plasmid

  • TG1 pUC18-cusRbox(2x)-GFP: same as TG1 pUC18-GFP, with two cusR-box sequences (part BBa_K1349002) inserted between the promoter and the GFP coding sequence.

  • TG1 pUC18-cusRbox(3x)-GFP: same as TG1 pUC18-GFP, with 3 cusR-box sequences (part BBa_K1349002) inserted between the promoter and the GFP coding sequence.

• 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.