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Part:BBa_K1139110:Experience

Designed by: Naoki Watarai   Group: iGEM13_Tokyo_Tech   (2013-09-09)

Pcon-LasR-Plux/tet-GFP

Materials & Methods

Our purpose is to check whether or not lux/tet hybrid promoter would be repressed when 3OC12HSL-LasR complex and protein TetR exist. We tried to compare the frequency of crosstalk in the presence or absence of the aTc, the TetR inhibitor.

We prepared six conditions as follow.

  • E-1) Culture containing crosstalk circumvention system cell with 3OC6HSL induction
  • E-2) Culture containing crosstalk circumvention system cell with 3OC12HSL induction
  • E-3) Culture containing crosstalk circumvention system cell with DMSO (no induction)

  • F-1) Culture containing crosstalk circumvention system cell with 3OC6HSL and aTc induction
  • F-2) Culture containing crosstalk circumvention system cell with 3OC12HSL and aTc induction
  • F-3) Culture containing crosstalk circumvention system cell with DMSO and aTc (no induction)

  • Positive control and negative control are similarly operated.

    • We also screened the concentration of 3OC6HSL and aTc, and examined activation of the crosstalk circumvention system circuit. Then we determined the transfer function of 3OC6HSL-LuxR complex by fitting Hill’s equation and introduced into our [http://2013.igem.org/Team:Tokyo_Tech/Modeling/Crosstalk_Circumvention modeling].


       1. Overnight Culture -> Fresh Culture -> Induction

    1.1 Prepare overnight culture of each cell (GFP posi, GFP nega, sample) at 37°C for 12 h.
    (=> O/N)

    1.2 Take 30 µL (from GFP posi, GFP nega, sample) of the overnight culture of inducer cell into
    LB (3 mL) + antibiotics (Amp 50 µg/mL+ Kan 30 µg/mL).
    (=> Fresh Culture)

    1.3 Incubate the flesh culture of cells (GFP posi, GFP nega, sample) until the observed OD600 reaches around 0.50.

    1.4a Take 30 µL each cell suspensions (GFP posi , GFP nega , sample) into

    LB (3 mL) + antibiotics (Amp 50 µg/mL + Kan 30 µg/mL) + 5 µM 3OC6HSL (3 µL),

    LB (3 mL) + antibiotics (Amp 50 µg/mL + Kan 30 µg/mL) + 5 µM 3OC6HSL (3 µL) + 0.05 µg/mL aTc (3 µL),

    LB (3 mL) + antibiotics (Amp 50 µg/mL + Kan 30 µg/mL) + 5 µM 3OC12HSL (3 µL),

    LB (3 mL) + antibiotics (Amp 50 µg/mL+ Kan 30 µg/mL) + 5 µM 3OC12HSL (3 µL)+ 0.05 mg/mL aTc (3 µL),

    LB (3 mL) + antibiotics (Amp 50 µg/mL + Kan 30 µg/mL) + 5 µM DMSO (3 µL),

    LB (3 mL) + antibiotics (Amp 50 µg/mL + Kan 30 µg/mL) + 5 µM DMSO (3 µL) + 0.05 mg/mL aTc (3 µL).

    1.4b Take 30 µL each cell suspensions (GFP posi , GFP

    nega , sample) into

    LB (3 mL) + antibiotics (Amp 50 µg/mL + Kan 30 µg/mL)+ C6 (3 µL) + aTc (3 µL) … inducer concentration for each sample is shown in below (Fig 3-2-3)


    aTc (µg/mL)




    C6 (nM)
    		00.050.150.51.55
    0No. 1No. 2No. 3No. 4No. 5No. 6
    0.3No. 7No. 8No. 9No. 10No. 11No. 12
    1No. 13No. 14No. 15No. 16No. 17No. 18
    3No. 19No. 20No. 21No. 22No. 23No. 24
    10No. 25No. 26No. 27No. 28No. 29No. 30
    30No. 31No. 32No. 33No. 34No. 35No. 36
    100No. 37No. 38No. 39No. 40No. 41No. 42
    Tab. 1. Inducer concentration for each sample

    1.5 Incubate all samples (6 samples X 6 kinds of culture = 36 samples) for another 4 h at 37°C.
    (=> Induction)

       2. Measurement (Flow cytometer)

    2.1 Measure all samples' OD600.

    2.2 Dilute all samples with 1X PBS to keep OD600 in the range from 0.2 to 0.5.

    2.3 Take 1 mL (from all samples) into disposable tube (for flow cytometer).

    2.4 Centrifuge them at 9,000g, 4°C, 1 min. and take their supernatant away.

    2.5 Suspend all samples with 1 mL 1X PBS.

    2.6 Measure all samples.

    2.7 Save and organize data.

    Result

    Fig. 1. Crosstalk circumvention results

    Fig. 1 shows the following. lux/tet hybrid promoter is repressed by TetR in the presence of 3OC6HSL-LuxR complex. Similarly, lux/tet hybrid promoter is repressed by TetR in the presence of 3OC12HSL-LasR complex. In brief, results from E-1) and F-1) are similar to those from E-2) and F-2). Therefore, the crosstalk circumvention experiment succeeded.

    Fig. 2. Activation of the crosstalk circumvention system circuit

    Fig. 2 shows activation of the crosstalk circumvention system circuit. It shows transition of the fluorescence intensity of GFP. It indicates that the concentration of C6 saturates between 10 mM and 30 mM.


    For more information, see [http://2013.igem.org/Team:Tokyo_Tech/Experiment/Crosstalk_Circumvention_Assay our work in Tokyo_Tech 2013 wiki].

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