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 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, S_cell, 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 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). The R.P.U. of a promoter is expressed as the ratio between its S_cell and the one of BBa_J23101:

This procedure has the advantage of being robust to variations in experimental conditions (for example the instrument used to measure absorbance and fluorescence) 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 to be constant on the time interval considered for R.P.U. evaluation.

Assaying 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 concentrations is useful to estimate the parameters of the activation Hill function of pLux promoter. In this way, for each of our tests, we were able to build a calibration curve for the biosensor:

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 (tuning of the dilution factor is necessary), you can easily evaluate the auto-inducer concentration as follows:

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