Team:UNIPV-Pavia/Measurements

From 2011.igem.org

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Revision as of 07:53, 17 September 2011

UNIPV TEAM 2011

Contents

Measuring promoters transcriptional strength

Measuring pTet transcriptional strength

  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

Measuring pLux transcriptional strength

  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 M
    • 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

Constitutive BBa_J23101-like promoters transcriptional strength

  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 six hours.
  4. 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

Measuring 3OC6-HSL synthesis and degradation

LuxI enzyme 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
    • 100 ng/ml
  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 200μl of supernatants (without resupsending the pelleted bacteria)
    • let the cultures grow at 37°C, 220 rpm until the next sampling
  5. Store supernatants at -20°C and measure 3OC6-HSL concentration according to the protocol based on BBa_T9002 biosensor.

AiiA enzyme 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 let them grow for two hours at 37°C, 220 rpm.
  3. Induce cultures with aTc; final concentrations:
    • 6 ng/ml
    • 8 ng/ml
    • 100 ng/ml
  4. Let the cultures grow for one more hour at 37°C, 220 rpm.
  5. Add 100 nM 3OC6-HSL.
  6. Collect supernatants (measuring the O.D. at 600 nm) at the moment of 3OC6-HSL addition, after 1 hour, 2 hours and 4 hours by:
    • take 250 μl of cultures
    • centrifuge them 13.300 rpm, 4 minutes
    • collect 200μl of supernatants (without resupsending the pelleted bacteria)
    • let the cultures grow at 37°C, 220 rpm until the next sampling
  7. Store supernatants at -20°C and measure 3OC6-HSL concentration according to the protocol based on BBa_T9002 biosensor.

3OC6-HSL degradation in M9 medium and cultures not expressing lactonases varying pH

  1. Inoculate 5 μl of long term glycerol stocks not expressing lactonases in 1 ml of M9 with the proper antibiotic. Use M9 at different pHs, for example pH = 6.0 and pH = 7.0. Let the cultures grow over night at 37°C, 220 rpm.
  2. Dilute cultures 1:100 in 4 ml M9 with the proper antibiotic in falcon tubes and let them grow for two hours at 37°C, 220 rpm.
  3. Prepare falcon tubes with M9 at pH = 6.0 and pH = 7.0.
  4. Add 100 nM 3OC6-HSL to each falcon tube.
  5. Collect supernatants (measuring the O.D. at 600 nm) at the moment of 3OC6-HSL addition, after 1 hour, 2 hours and 4 hours by:
    • take 250 μl of cultures
    • centrifuge them 13.300 rpm, 4 minutes
    • collect 200μl of supernatants (without resupsending the pelleted bacteria)
    • let the cultures grow at 37°C, 220 rpm until the next sampling
  6. Store supernatants at -20°C and measure 3OC6-HSL concentration according the protocol based on BBa_T9002 biosensor.

Measuring 3OC6-HSL concentration with BBa_T9002

  1. Inoculate 5 μl BBa_T9002 in 1 ml M9 with the proper antibiotic (Ampicillin when you use BBa_T9002 or Ampicillin + Chloramphenicol 12.5 mg/ml if you use T9002-ENTERO, see Freezer Management) together with a non-fluorescent culture; let them grow over night at 37°C, 220 rpm.
  2. Dilute cultures 1:100 in M9 with the proper antibiotic; let the cultures grow 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 the wells of the microplate.
  4. Don't forget to build a calibration curve, by inducing BBa_T9002 cultures with known 3OC6-HSL concentrations:
    • 0 M
    • 0.1 nM
    • 0.2 nM
    • 0.5 nM
    • 1 nM
    • 2 nM
    • 5 nM
    • 10 nM
    • 100 nM
    • 1 μM
  5. Use Tecan Infinite F200 to read O.D. at 600 nm and green fluorescence, 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

Data analysis

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