Team:UNIPV-Pavia/Parts/Characterization

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

Data analysis for self-inducible promoters (initiation-treshold determination)

Data analysis for RPU evaluation