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Contents 1 Time-delay check 1.1 Purpose 1.2 Equipments　and Materials 1.2.1 Equipment 1.2.2 Materials 1.3 Protocol 1.3.1 Culturing overnight (before the testing day): 1.3.2 Following day 1.3.3 Measurement

Time-delay check

Purpose

To Confirm that communication using non-endogenous molecules results in slower activation of receivers.

Equipments　and Materials

Equipment

• shaking incubator(37°C,30°C)
  • Innova 4200 Benchtop or Floor-Stackable Incubator Shaker(37°C)
  • Taitec BioShaker BR-33FM(30°C,200rpm)
• 46-well plate(deep well)
• Fluoroskan Ascent 2.5(program:Ascent Software Version 2.6)
• Beckman Allegra^tm X-12R Centrifuga(Beckman Coulter)

Materials

• AHL(100μM)
• E.coli Culture Containing BBa_T9002
• E.coli Culture Containing plasmids you will testing

Protocol

Culturing overnight (before the testing day):

1. Inoculate all cultures you will testing from gloycerol stocks into 2 mL of LB-ampicillin liquid medium.
2. Also inoculate a culture containing BBa_T9002 into 2 mL of LB-ampicillin liquid medium.
3. Incubate all cultures with shaking at 37°C(O/N).

Following day

• cultures containing BBa_T9002

1. Inoculate a culture by adding 100 μL of the cultures into 40mL of LB-ampicillin medium.(in a flask).
2. Incubate a culture for 6-8 hours with shaking at 37°C.
3. Wash
   1. Aliquote 10mL of the culture into 50 mL four 50 mL falcon tubes.
   2. Cultures are centrifuged at 3500 rpm for 6 minutes.
   3. Dinspense supernatant.
   4. Add 10mL of new LB-ampicillin medium and resuspense with pipetting.
4. aliquote 1mL of the cultures into a 48-deep well plate(deep well).

• cultures containing plasmids you will testing

1. Inoculate cultures by adding 12.5 μL of the cultures into 5 mL of LB-ampicillin medium.
2. Incubate cultures for 6-8 hours with shaking at 37°C.
3. Wash
   1. Cultures are centrifuged at 3500　rpm for 6 minutes.
   2. Dinspense supernatant.
   3. Add 3mL of new LB-ampicillin medium and resuspense with pipetting.
4. repeat washing process twice.
5. Cultures are centrifuged at 3500　rpm for 6 minutes.
6. Dinspense supernatant.
7. Add 5　mL of new LB-ampicillin medium and resuspense with pipetting.
8. aliquote cultures into a 48-deep well plate(deep well).

Measurement

• Mix senders and receiver as shown in Table below:

Senders culture(μL)	BBa_T9002 culture(μL)	Receiver cells/Sender cells
500	500	1
100	1000	10
10	1000	100

• Prepare three replicate cultures.

1. Incubate testing cultures with shaking at 37°C.
2. After some intervals,aliqupte 100 μL of testing cultures into a 96-well plate(shallow well).
3. Measure fluorescence intensity.

• conditions
  • shaking(before measurement):On time = 1min,Off time = 10 sec,
  • integration time = 1000 ms
  • Beam width:Normal Beam
  • Wavelength pair = 485 nm(excitation) and 527 nm(emission)

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