Team:UNIPV-Pavia/Measurements

pTet protocol

1. Streak long term storage glycerol stocks on a LB agar plate + Cm12.5 (don't forget positive and negative controls). Let them grow over night.
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.
3. Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow 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). Grow cultures for three hours.
5. 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 (don't forget positive and negative controls). Let them grow over night.
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.
3. Dilute cultures 1:500 in 1 ml of M9 + Cm12.5 and let them grow for three hours.
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). Grow cultures for three hours.
5. 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 grow the cultures over night.
2. Dilute cultures 1:100 in 4 ml M9 + Cm12.5 in falcon tubes and grow them for two hours.
3. Induce cultures with aTc (final concentrations: 6 ng/ml, 8 ng/ml and 100 ng/ml) and wait for one hour.
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)
6. Store supernatants at -20°C.
7. Inoculate 5 μl BBa_T9002 in 1 ml M9 (together with a negative control culture); let them grow over night.
8. Dilute BBa_T9002 and negative control 1:100 in M9; grow cultures for two hours.
9. 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
   • RFP filters: 485 nm (excitation) / 540 nm (emission)
   • duration time: 10 - 15 hours

LuxI activity

1. Inoculate 5 μl of long term glycerol stocks in 1 ml of M9 + Cm12.5 and grow the cultures over night.
2. Dilute cultures 1:100 in 4 ml M9 + Cm12.5 in falcon tubes and grow them for two hours.
3. Induce cultures with aTc (final concentrations: 6 ng/ml, 8 ng/ml and 100 ng/ml) and wait for one hour.
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)
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.
7. Dilute BBa_T9002 and negative control 1:100 in M9; grow cultures for two hours.
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
   • RFP filters: 485 nm (excitation) / 540 nm (emission)
   • duration time: 10 - 15 hours