Team:UNIPV-Pavia/Measurements

pTet protocol

1. Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.
2. Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37 °C, 220 rpm.
3. Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow at 37°C, 220 rpm for three hours.
4. Induce cultures in falcon tube with anhydrotetracycline (aTc); final concentrations:
   • 0 ng/ml
   • 1 ng/ml
   • 2 ng/ml
   • 3 ng/ml
   • 4 ng/ml
   • 5 ng/ml
   • 8 ng/ml
   • 10 ng/ml
   • 50 ng/ml
   • 100 ng/ml
5. Let the cultures grow at 37°C, 220 rpm for three hours.
6. Aliquot 200 μl of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:
   • temperature: 37°C
   • sampling time: 5 minutes
   • 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements
   • fluorescence gain: 50 - 80
   • O.D. filter: 600 nm
   • RFP filters: 535 nm (excitation) / 620 nm (emission)
   • duration time: 10 - 15 hours

pLux protocol

1. Streak long term storage glycerol stocks on a LB agar plate + Cm12.5, in order to have single colonies (don't forget positive and negative controls). Let them grow over night at 37°C.
2. Pick 3 colonies from each clone and inoculate it in 1 ml M9 + Cm12.5 in a falcon tube; let them grow over night at 37°C, 220 rpm.
3. Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow for three hours at 37°C, 220 rpm.
4. Induce cultures in falcon tube with 3OC₆-HSL; final concentrations:
   • 0 nM
   • 0.1 nM
   • 0.5 nM
   • 1 nM
   • 2 nM
   • 5 nM
   • 10 nM
   • 100 nM
5. Let the cultures grow for three hours at 37°C, 220 rpm.
6. Aliquot 200 μl of cultures in microplate wells and measure O.D. and fluorescence with Tecan Infinite F200 microplate reader. Set the automatic procedure:
   • temperature: 37°C
   • sampling time: 5 minutes
   • 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements
   • fluorescence gain: 50 - 80
   • O.D. filter: 600 nm
   • RFP filters: 535 nm (excitation) / 620 nm (emission)
   • duration time: 10 - 15 hours

Enzyme activity assay

AiiA activity

1. Inoculate 5 μl of long term glycerol stocks in 1 ml of M9 + Cm12.5 and let the cultures grow over night at 37°C, 220 rpm.
2. Dilute cultures 1:100 in 4 ml M9 + Cm12.5 in falcon tubes and grow them for two hours at 37°C, 220 rpm.
3. Induce cultures with aTc (final concentrations: 6 ng/ml, 8 ng/ml and 100 ng/ml) and wait for one hour at 37°C, 220 rpm.
4. Add 100 nM 3OC₆-HSL.
5. Collect supernatants (measuring the O.D. at 600 nm) at the moment of induction, after 1 hour, 2 hours and 4 hours by:
   • take 250 μl of cultures
   • centrifuge them 13.300 rpm, 4 minutes
   • collect the supernatants (without resupsending the pelleted bacteria)
   • grow cultures at 37°C, 220 rpm
6. Store supernatants at -20°C and measure 3OC₆-HSL concentration using BBa_T9002 according to 3OC₆-HSL concentration protocol.

   LuxI activity

   1. Inoculate 5 μl of long term glycerol stocks in 1 ml of M9 + Cm12.5 and let the cultures grow over night at 37°C, 220 rpm.
   2. Dilute cultures 1:100 in 4 ml M9 + Cm12.5 in falcon tubes and grow them for two hours at 37°C, 220 rpm.
   3. Induce cultures with aTc (final concentrations: 6 ng/ml, 8 ng/ml and 100 ng/ml) and wait for one hour at 37°C, 220 rpm.
   4. Collect supernatants (measuring the O.D. at 600 nm) at the moment of induction, after 1 hour, 2 hours and 4 hours by:
      • take 250 μl of cultures
      • centrifuge them 13.300 rpm, 4 minutes
      • collect the supernatants (without resupsending the pelleted bacteria)
      • grow cultures at 37°C, 220 rpm
   5. Store supernatants at -20°C.
   6. Inoculate 5 μl BBa_T9002 in 1 ml M9 (together with a negative control culture); let them grow over night at 37°C, 220 rpm.
   7. Dilute BBa_T9002 and negative control 1:100 in M9; grow cultures for two hours at 37°C, 220 rpm.
   8. Measure 3OC₆-HSL concentration of the previously collected supernatants (diluting them 1:20), inducing BBa_T9002 cultures: aliquot 190μl of inducible cultures and 10 μl of supernatants in each well of the microplate. Don't forget to build a calibration curve, by inducing BBa_T9002 cultures with known 3OC₆-HSL concentrations (0 nM, 0.1 nM, 0.2 nM, 0.5 nM, 1 nM, 2 nM, 5 nM, 10 nM, 100 nM, 1 μM). Use Tecan Infinite F200, setting the automatic procedure:
      • temperature: 37°C
      • sampling time: 5 minutes
      • 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements
      • fluorescence gain: 50
      • O.D. filter: 600 nm
      • GFP filters: 485 nm (excitation) / 540 nm (emission)
      • duration time: 10 - 15 hours

   3OC₆-HSL concentration protocol

   1. Inoculate 5 μl BBa_T9002 in 1 ml M9 (together with a negative control culture); let them grow over night at 37°C, 220 rpm.
   2. Dilute BBa_T9002 and negative control 1:100 in M9; grow cultures for two hours at 37°C, 220 rpm.
   3. Measure 3OC₆-HSL concentration of the previously collected supernatants (diluting them 1:20), inducing BBa_T9002 cultures: aliquot 190μl of inducible cultures and 10 μl of supernatants in each well of the microplate. Don't forget to build a calibration curve, by inducing BBa_T9002 cultures with known 3OC₆-HSL concentrations (0 nM, 0.1 nM, 0.2 nM, 0.5 nM, 1 nM, 2 nM, 5 nM, 10 nM, 100 nM, 1 μM). Use Tecan Infinite F200, setting the automatic procedure:
      • temperature: 37°C
      • sampling time: 5 minutes
      • 15 seconds of linear shaking (3 mm amplitude) followed by 5 seconds waiting before measurements
      • fluorescence gain: 50
      • O.D. filter: 600 nm
      • GFP filters: 485 nm (excitation) / 540 nm (emission)
      • duration time: 10 - 15 hours