IGEM:IMPERIAL/2007/Experimental Design/Phase1/Protocol 1.2

Protocol

Day 1

Equipment

• Incubator 37^oC
• Stripettes
• Gilson Pipettes
• Sterile conical flasks x4

Reagents

• Different strains of expression hosts with the desirable plasmid x4
• LB medium
• Ampicillin stock (50 mg/ml)

Innoculation

• Make 3 x 100ml LB medium with 100µl ampicillin and 1 x 250ml LB medium with 250µl ampicillin into sterile concical flasks.
• Innoculate the three cultures into the the conical flasks
• Leave overnight at 37°C with shaking

Day 2

Equipment

• Incubator 30^oC
• Fluorometer
• Well-plate x1
• Plate Centrifuge
• 6x1.5ml tubes
• Gilson Pipettes
• Stop watch

Equipment

• Incubator 30^oC
• Fluorometer
• Spectrometer
• Plate x1
• Plate Centrifuge
• 6x1.5ml tubes
• Gilson Pipettes
• Stop watch

Day 3:

Make 5 of 550 ml LB medium with 550 µl ampicillin in sterile conical flask Innoculate in 5 cultures into the the conical flasks Leave overnight at 37°C with shaking

Day 4:

Prepare a water bath at 30°C [should we use a shaking incubator?] Aliquot the 550 ml cultures into sterile tubes, 25 ml per tube Add 53.3075 ng AHL each time to increase [AHL] in steps of 10nM Begin the timer when AHL is added At 5 minute intervals for the first 40 minutes, and 10 minute intervals thereafter: Sample a 1 ml of the reaction mixture Immediately chill in ice (to slow the rate of reaction) Centrifuge at maximum speed for 30 seconds to pellet the cells Remove the supernatant and resuspend the pellet in 1ml distilled water Put in a spectrometer and excite it at 501nm and detect at 511nm [Note: Use solution at 0 minutes as a blank, assume no fluorescence is present prior to activation] These measurements should be repeated with the other 4 cultures of bacteria