Team:UNIPV-Pavia/Measurements

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 withBBa_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, S_cell, or Relative Promoter Unit, R.P.U.) blanking is needed:

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

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 doubling time can be evaluated as:

When measuring multiple growth for a strain this value was evaluated for each reaplicate and then average was computed.

Synthesis rate per cell (S_cell) evaluation

Another interesting parameter, which describes the activity of a promoter of interest (φ), is the S_cell; it needs to be measured using reporter genes as RFP or GFP.
Compute the average of the time derivative of the red or green fluorescence, divided by the O.D.₆₀₀, in the time interval corresponding to the exponential growth phase which boundaries can be identified by visual inspection of the logarithmic growth curve:

The S_cell is basic to measure the Relative Promoter Unit, a robust parameter expressing the transcriptional activity of a promoter.

R.P.U. evaluation

As described in Kelly J. et al., 2009, Relative Promoter Units express the activity of a promoter of interest (φ), reported to the one of a reference promoter, BBa_J23101 (from Anderson promoters collection):

This procedure has the advantage of being robust to variations in experimental conditions and proportional to PoPS (Polymerase Per Second) if the subsequent specifications are satisfied:

• strain, plasmid copy number, antibiotic, growth medium, growth conditions, protein generator assembled downstream of the promoter must be the same in the promoter of interest and in BBa_J23101 reference standard
• the reporter protein must have a half life higher than the experiment duration

Moreover the S_cell signal of both the promoter of interest and of BBa_J23101 has tp be at the steady state.

Extrapolating 3OC₆-HSL concentration

The procedure described below is useful in order to quantify the concentration of 3OC₆-HSL in cultures expressing LuxI and AiiA enzyme.
Once collected (according to the protocol above) and pre-processed data, it is necessary to compute the S_cell of every culture.
BBa_T9002 induced with known 3OC₆-HSL concentration is useful to estimate the parameters of the activation Hill function of pLux promoter:

Provided that the S_cell measured for BBa_T9002 induced with supernatants of cultures producing or degrading 3OC₆-HSL has a value included in the linear zone of the biosensor, you can easily extrapolate the auto-inducer concentration as follows:

Finally, don't forget to multiply this value for the dilution factor (in our experiments it was 20).