Team:UNIPV-Pavia/Measurements

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UNIPV TEAM 2011

Measurements

Contents

NB: unless differently specified, all tests were performed in E. coli MGZ1 in M9 supplemented medium at 37°C.

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_J23101x 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. Induce BBa_T9002 cultures with the previously collected supernatants, diluting them 1:20: 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

Data pre-processing

Data from TECAN Infinite F200 need to be pre-processed in order to remove spurious effects affecting measurements. Before evaluating the parameters of interest (for example the synthesis rate per cell, Scell, or Relative Promoter Unit, R.P.U.) blanking is needed:

    • the reference value (i.e. the measure of O.D. at 600 nm of the broth in which cultures grow) was subtracted from each sample of O.D. at 600 nm, (O.D.600)
    • the reference value (i.e. the red or green fluorescence of a non-fluorescent culture, also known as auto-fluorescence) was also subtracted from each sample of red and green fluorescence

Doubling time evaluation

After blanking O.D.600 data you can compute the doubling time of cultures growth, i.e. the time needed to double O.D.600.
Identifying the exponential phase of growth curve can be done by visual inspection, plotting the natural logarithm of O.D.600 over time. Linear regression on logarithmic data is performed to estimate the growth rate, named μ. Finally d

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