Microplate reader experiments for self-inducible promoters
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)
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 for self-inducible promoters (initiation-treshold determination)
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