Team:Technion HS Israel/Experiments

Technion 2015 HS Team's Wiki

Experiments

To test our genetic circuit and to be more certain about it, we started testing the first part of our construct with and without Aiia by measuring the florescence of the YFP that replaced the tetR (check the parts ) , after that we test our whole construct with and without Aiia by measuring the florescence of the RFP that replaced the CCDB (check the parts ) the results that we got are found in the results page.

The experimental setup is as follows:

An overnight culture of the bacteria harboring the genetic circuit (based on LuxR (BBa_C0062), AiiA (BBa_C0060), TetR (BBa_R0040) and a fluorescent protein) will be diluted (1:100) and incubated with increasing concentration of 3OC6-HSL (sigma: K3007, max conc: 10000nM). Measurements of fluorescent protein expression will be conducted using a microplate reader. The protocol is provided down bellow .

The protocol

AHL inducer experiment protocol

  • 1. Purpose:

    test the response of our bio brick to AHL exposure measuring its fluorescence according to a range of AHL concentrations and determining whether AiiA functions properly.
  • 2. Materials:
    • - AHL
    • - Bacteria samples:
      • 2x (+)AiiA
      • 2x (-)AiiA
    • - 2x Eppendorf
    • - LB
    • - Chloramphenicol
  • 3. Methods:

    Prepare 5 ml starter by growing the cells in 5ml LB medium + appropriate antibiotics (5 µl of chloramphenicol, CM, ) at 37°C overnight.

    Samples:
    • (1) +AiiA (K176003)
    • (2) – AiiA (K176005)
    The next day:

    • 1- Prepare BA+5% (v/v) LB
      • a. 47.5 ml of BA plus 2.5 ml of LB
      • b. Add appropriate antibiotics (chloramphenicol: 50 µl)
    • 2- dilute bacteria 1:100


      • Add 20 µl of bacteria into 2 ml of (BA+5% LB) in a 48 well plate

    • 3- Add AHL(3OC8) in the 48 well plate in proper concentrations.
    • 4- Incubate it for 2 hours in a shaker ( 37°C, 250 RPM). After 2 hours, measure the absorbance (OD600 – for cell concentration) and fluorescence (excitation peak: 584nm, emission peak: 608nm) using a plate reader. A measurement should be taken every 30 minutes for 3.5 hours (to a total of 7 times).


    48 well plate AHL(3OC8) concentration preparation:

    1 2 3 4 5 6 7 8
    mL 2.2 2 2 2 2 2 2 2
    AHL(3OC8) nM 10000 100 100 10 1 0.1 0.01 0
    AHL(3OC8) µM 10 1 0.1 0.010 0.001 0.0001 0.00001 0
    STEPS
    1 add 0.55 µL
    2 Take 0.2ml Add here
    3 Take 0.2ml Add here
    4 Take 0.2ml Add here
    5 Take 0.2ml Add here
    6 Take 0.2ml Add here
    7 Take 0.2ml Add here
    8 Take 0.2ml
    9 Add 0.19995ml (bacteria with BA and lb)and 0.5µl AHL(3OC8)