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August/18 October 2009
From 2009.igem.org
(New page: TEST : sencer check<br> Osaka team experiment FINISH! [https://2009.igem.org/Team:Osaka/NOTES back to NOTES]) |
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| - | + | Receivers-AHL 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 - Measurement | ||
| + | 1. Transfer 1ml of the overnight culture into 30ml fresh LB culture medium. Incubate in shaking incubator at 37 degrees. | ||
| + | |||
| + | 2. Measure OD of culture every hour or so, until OD reaches fixed value (culture at full growth). | ||
| + | |||
| + | 3. Transfer culture into 50ml ultracentrifuge tube and spin at 8000rpm for 5 minutes. | ||
| + | |||
| + | 4. Resuspend pellet with 30ml fresh LB culture. | ||
| + | |||
| + | 5. Incubate culture in shaking incubator at 30 degree Celcius for 10 minutes. | ||
| + | |||
| + | 6. Transfer 1ml of culture into microcentrifuge tube and put on ice. Immediately add AHL to rest of culture to attain desired concentration. | ||
| + | |||
| + | (Note: Between the steps detailed below, samples should be kept on ice to minimize cellular activity.) | ||
| + | |||
| + | 7. Obtain OD600 of 2x diluted sample by transferring 500ul of sample into cuvette, adding 500ul MiliQ water and measuring the resulting OD. | ||
| + | |||
| + | 8. Ultracentrifuge remaining 500ul of sample at 13000rpm for 2min, discard supernatant, then resuspend pellet with 1ml MiliQ water. | ||
| + | |||
| + | 9. Ultracentrifuge sample again at 13000rpm for 2min, discard supernatant and resuspend with precisely 1ml MiliQ water (this results in 2x dilution). | ||
| + | |||
| + | 10. Measure GFP fluorescence with a fluorimeter at 488nm excitation, 513nm excitation. | ||
| + | |||
| + | 11. Every hour, sample 1ml of culture and repeat steps 7-10 until fluorescence reaches steady state. | ||
| + | |||
| + | |||
| + | Comments: | ||
| + | It was realized sometime after starting this experiment that in order to measure efficiency of promoter activation, the rate of change of GFP fluorescence would have been more useful than the absolute value of GFP fluorescence itself. Also, promoter activity might peak somewhere between the addition of AHL and the first subsequent measurement as response times of <10min has been reported for similar devices. The solution would have been to take measurements on smaller time intervals, eg. every minute, and calculate rate of change of fluorescence from the measurements. Unfortunately we were not able to access instruments capable of automatically measuring fluorescence at smaller time intervals. | ||
Revision as of 10:21, 21 October 2009
Receivers-AHL 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 - Measurement 1. Transfer 1ml of the overnight culture into 30ml fresh LB culture medium. Incubate in shaking incubator at 37 degrees.
2. Measure OD of culture every hour or so, until OD reaches fixed value (culture at full growth).
3. Transfer culture into 50ml ultracentrifuge tube and spin at 8000rpm for 5 minutes.
4. Resuspend pellet with 30ml fresh LB culture.
5. Incubate culture in shaking incubator at 30 degree Celcius for 10 minutes.
6. Transfer 1ml of culture into microcentrifuge tube and put on ice. Immediately add AHL to rest of culture to attain desired concentration.
(Note: Between the steps detailed below, samples should be kept on ice to minimize cellular activity.)
7. Obtain OD600 of 2x diluted sample by transferring 500ul of sample into cuvette, adding 500ul MiliQ water and measuring the resulting OD.
8. Ultracentrifuge remaining 500ul of sample at 13000rpm for 2min, discard supernatant, then resuspend pellet with 1ml MiliQ water.
9. Ultracentrifuge sample again at 13000rpm for 2min, discard supernatant and resuspend with precisely 1ml MiliQ water (this results in 2x dilution).
10. Measure GFP fluorescence with a fluorimeter at 488nm excitation, 513nm excitation.
11. Every hour, sample 1ml of culture and repeat steps 7-10 until fluorescence reaches steady state.
Comments:
It was realized sometime after starting this experiment that in order to measure efficiency of promoter activation, the rate of change of GFP fluorescence would have been more useful than the absolute value of GFP fluorescence itself. Also, promoter activity might peak somewhere between the addition of AHL and the first subsequent measurement as response times of <10min has been reported for similar devices. The solution would have been to take measurements on smaller time intervals, eg. every minute, and calculate rate of change of fluorescence from the measurements. Unfortunately we were not able to access instruments capable of automatically measuring fluorescence at smaller time intervals.
