The BioBricks Foundation:Standards/Technical/Measurement/Promoter characterization experiment (FACS)

FACS protocol V0.1

Materials

• 5 strains provided as stabs from Endy Lab
  • TOP10,<bbpart>I20259</bbpart>,<bbpart>I20260</bbpart>,<bbpart>I20268</bbpart>,<bbpart>I20269</bbpart>,<bbpart>I20270</bbpart>
• TOP10 background strain (Jason will provide so we're all actually using the same one).
• 100 mM sodium phosphate, 150 mM sodium chloride, pH 7.4 (PBS)
• 5 mL polystyrene round bottom tube with cell strainer cap: BD Falcon #352235
  • Jason R. Kelly 13:37, 15 February 2008 (EST):What's the cell strainer cap for? Don't normally use that.

Culturing of the strains

1. Streak LB+Kan plates of the 5 test strains and an LB (no antibiotic) for TOP10
2. For each test construct add 5ml of supplemented M9 medium (w/glycerol, see preparation protocol) and Kanamycin (20ug/ml) to X 17mm test tubes. Add 5ml of supplemented M9 medium (w/glycerol) with no antibiotic for the TOP10 negative control cells to X 17mm test tubes.
   • how many replicates do we want to do (X)?
3. Inoculate media by picking single colonies from plates. Grow cultures for 20 hrs at 37°C with spinning at 70 rpm.

1. Grow an overnight culture (20hrs) in 5ml tubes on roller in supplemented M9 @ 37C 2. In the morning dilute 100x into 5ml to get cells back into Log phase let grow for 4hrs 3. Take OD600 and then dilute cultures to OD 0.07 in 5ml media (pre-warmed to 37C) 4. Let grow for 1hr should be at an OD of 0.1 - 0.15, take cells and put immediately on ice 5. spin down and resuspend in PBS and then you can run on flow

1. After 20 hrs, cultures were diluted 1:100 into 5 ml of pre-warmed fresh media and grown for approximately four hours under the same conditions(17mm tubes, warm room, spinning at 70 rpm).
2. After four hours, a 500ul aliquot from each culture was then used for OD600 measurement. The cultures were diluted to OD 0.07 in 5 ml of pre-warmed fresh media to be grown for one hour under the same conditions(17mm tubes, warm room, spinning at 70 rpm).
3. For each culture a 200 µl aliquot was transferred into a flat-bottomed 96 well plate (Cellstar Uclear bottom, Greiner). An additional control of blank media was also added in the same aliquot. Three replicates (three aliquots of 200 ul) of each culture were measured.
4. The plate was incubated in a Wallac Victor3 multi-well fluorimeter (Perkin Elmer) at 37°C and assayed with an automatically repeating protocol of absorbance measurements (600 nm absorbance filter, 0.1 second counting time through 5 mm of fluid), fluorescence measurements (488 nm excitation filter, 525 nm emission filter, 0.5 seconds, CW lamp energy 12902 units), and shaking (1 mm, linear, normal speed, 5 seconds). Time between repeated measurements for each well was 2 min and 21 s.
5. Data processing was used to calculate the GFP synthesis rates for each well. The data for each construct tested was averaged across the three replicates wells and the three replicate cultures.

Live Cell Procedure

• When your 5 mL culture reaches OD600 = 0.100, pellet the cells by centrifuging at 3000xg at room temperature for 5 minutes.
• Remove the supernatant.
• Resuspend the cell pellet in 5 mL PBS.
• Re-pellet the cells by centrifuging at 3000xg at room temp for 5 min.
• Repeat wash with 5 mL PBS.
• Re-suspend washed cells in 0.5 mL PBS. The cell concentration should be approximately 5x10⁸ cells/mL.
• Add 250 uL washed cells through the cell strainer lid into a 5 mL polystyrene tube. To get the solution to pass through the strainer, apply slight pressure to the strainer lid with the pipet tip as dispensing the cell solution.
• Add 250 uL PBS through the cell strainer lid into the 5 mL polystyrene tube.
• Put cells on ice.
• Analyze cells by flow cytometry using 488 nm excitation as quickly as possible.