Team:UNIPV-Pavia/Parts/Characterization

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CHARACTERIZATION

Growth conditions

Microplate reader experiments for self-inducible promoters - Protocol #1

8 ul of long term storage glycerol stock were inoculated in 1 ml of LB or M9 + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours.
The grown cultures were then diluted 1:100 in 1 ml of LB or M9 supplemented medium and incubated in the same conditions as before for about 4 hours.
These new cultures were further grown for 4 hours (37 °C, 220 rpm) and then pelletted (2000 rpm, 10 minutes) in order to eliminate the HSL produced during the first growth.
Supernatants were discarded and the pellets were resupsended in 1ml LB or M9 + suitable antibiotic and transferred to 1,5 ml Eppendorf tube
Immediately, these cultures were diluted 1:1000 (1ul in 1ml LB or M9 + suitable antibiotic) and aliquoted in a flat-bottom 96-well microplate in triplicate, avoiding to perform dynamic experiments in the microplate frame (see Frame effect section – past year for details). All the wells were filled with a 200 ul volume.
The microplate was incubated in the Tecan Infinite F200 microplate reader and fluorescence (when required) and absorbance were measured with this automatic protocol:
- 37°C constant for all the experiment;
- sampling time of 5 minutes;
- fluorescence gain of 50;
- O.D. filter was 600 nm;
- GFP filters were 485nm (ex) / 540nm (em);
- 15 seconds of linear shaking (3mm amplitude) followed by 10 seconds of waiting before the measurements in order to make a homogeneous culture.
- Variable experiment duration time (from 3 to 24 hours).

Microplate reader experiments for constitutive promoters (R.P.U. evaluation) - Protocol #2

8 ul of long term storage glycerol stock were inoculated in 5 ml of LB or M9 + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours.
The grown cultures were then diluted 1:100 in 5 ml of LB or M9 supplemented medium and incubated in the same conditions as before for about 4 hours.
These new cultures were diluted to an O.D.600 of 0.02 (measured with a TECAN F200 microplate reader on a 200 ul of volume per well; it is not comparable with the 1 cm pathlength cuvette) in 2ml LB or M9 + suitable antibiotic.
These new dilutions were aliquoted in a flat-bottom 96-well microplate, avoiding to perform dynamic experiments in the microplate frame (see Frame effect section for details). All the wells were filled with a 200 ul volume.
The microplate was incubated in the Tecan Infinite F200 microplate reader and fluorescence (when required) and absorbance were measured with this automatic protocol:
- 37°C constant for all the experiment;
- sampling time of 5 minutes;
- fluorescence gain of 50;
- O.D. filter was 600 nm;
- GFP filters were 485nm (ex) / 540nm (em);
- 15 seconds of linear shaking (3mm amplitude) followed by 10 seconds of waiting before the measurements in order to make a homogeneous culture.
- Experiment duration time (about 6 hours).

Data Analysis

Preliminary remarks

All our growth curves have been obtained subtracting for each time sample the broth O.D.600 measurement from that of the culture; broth was considered in the same conditions of the culture (e.g. induced with the same inducer concentration and supplemented with the same antibiotic of the culture).

Doubling time evaluation

The natural logarithm of the growth curves (processed according to the above section) was computed and the linear phase (corresponding to the bacterial exponential growth phase) was isolated by visual inspection. Then the linear regression was performed in order to estimate the slope of the line m. Finally the doubling time was estimated as d=ln(2)/m [minutes].

In the case of multiple growth curves for a strain, the mean value of the processed curves was computed for each time sample before applying the above described procedure.

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 ===Data analysis for RPU evaluation===
-The RPUs are standard units proposed by  Kelly J. et al., 2008, in which the transcriptional strength of a promoter can be measured using a reference standard.
-RPUs have been computed as:
-in which:
-    * phi is the considered promoter and J23101 is the reference standard promoter (taken from Anderson Promoter Collection);
-    * F is the blanked fluorescence of the culture, computed subtracting for each time sample fluorescence measure for negative control from that of culture, where the negative control is a non-fluorescent strain (in our experiment it is usually used TOP10 strain bearing BBa_B0032 or BBa_B0033, which are symmply RBSs do not have expression systems for reporter genes);
-    * ABS is the blanked absorbance (O.D.600) of the culture, computed as described in "Growth curves" section.
-RPU measurement has the following advantages (under suitable conditions)
-    * it is proportional to PoPS (Polymerase Per Second), a very important parameter that expresses the transcription rate of a promoter;
-    * it uses a reference standard and so measurements can be compared between different laboratories.
-The hypotheses on which RPU theory is based can be found in Kelly J. et al., 2008, as well as all the mathematical steps. From our point of view, the main hypotheses that have to be satisfied are the following:
-    * the reporter protein must have a half life higher than the experiment duration (we use GFPmut3, BBa_E0240, which has an estimated half life of at least 24 hours, and the experiments duration is always less than 7 hours);
-    * 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 J23101 reference standard.
-    * steady state must be valid, so (dF/dt)/ABS (proportional to the GFP synthesis rate per cell) must be constant.