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August/20 October 2009
From 2009.igem.org
Sender-Receiver Test
Protocol:
Day 1 - Preparation
1. From transformation plate, pick up a small quantity of cells from a colony using sterilized toothpick and streak on LA plate with the appropriate antibiotic resistance. Incubate at 37 degrees Celsius for 8+ hr.
2. From the streak plate, choose a single-cell colony and pick up cells using sterilized toothpick, then inoculate 5ml LB culture medium to which the appropriate antibiotic resistance (50mg/l) has been added. Incubate overnight at 37 degrees Celsius in a shaking incubator.
Day 2 - Mix & Culture
1. Prepare microcentrifuge tubes: 3 samples for each sender-receiver pair (sender-receiver test) or AHL concentration (receiver-only test). Add 400ul of LB-Amp culture medium to each microcentrifuge tube.
2. Add AHL to each microcentrifuge tube that will be used for receiver test.
3. Transfer 1ml of each overnight culture to separate microcentrifuge tubes and ultracentrifuge at 12000rpm for 1min. Discard supernatant and resuspend cells with 1ml LB-Amp. Repeat with remaining overnight culture if necessary to obtain the required amount of cell culture.
4. For sender-receiver test: Add 50ul each of sender and receiver cultures to a microcentrifuge tube prepared in step 1. For receiver-only test: Add 100ul of receiver culture to a microcentrifuge tube that has been prepared in steps 1 & 2 (AHL added).
5. Incubate microcentrifuge tubes at 37 degrees Celsius overnight.
Day 3 - Observation & Measurement
1. Wash cells by ultracentrifuging at 13000rpm for 2min, discarding supernatant and resuspending cells with MiliQ water. Repeat, and resuspend with precisely 1ml MiliQ water.
2. For direct observation, bring cells suspended in water to darkened room and shine with UV Black Light. GFP fluorescence should be observable if sufficiently strong promoter activation has been achieved.
3. Measure GFP fluorescence with a fluorimeter at 488nm excitation, 513nm excitation.
Comments:
3 samples were prepared for each set of conditions (sender-receiver pairing or AHL concentration-receiver pairing). This was done in order to obtain averages for more accurate results. However it was observed that the 3 measurement values for each set of condition often showed great deviation from each other. This can be attributed to both human error and instrumental error, both of which were likely amplified by the small volume of sample used.
Therefore, this experiment is not recommended for quantitative characterization of sensor parts. It does serve, to a limited extent, as a qualitative evaluation of which pairings work and which don't, which enabled us to determine which of our devices were actually able to function as planned.
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