Endy:F2620/Stability/Protocols

1. Inoculate two 5ml cultures of Supplemented M9 medium and antibiotic (kanamycin, 20μg/ml) with a single colony from a fresh plate. Grow the culture in a 17mm test tube for 15hrs at 37C with shaking at 70rpm. One culture is supplemented with a high input level of AHL (100nM) added to it.
2. Cultures were diluted 1:400 into fresh medium. The saturating concentration of AHL in one culture was maintained. Cultures were further grown under the same conditions for an additional 10h. A 1:4096 dilution was performed and the saturating concentration of AHL in one culture was maintained. The cultures were then allowed to grow overnight. This propagation was contained for 5 days.
3. Each day, following the overnight incubation of cultures, bulk plasmid DNA from each culture was purified using a Qiagen Spin Miniprep Kit. That DNA was sequenced at the Biopolymers Lab at MIT using primers internal and external to the receiver and reporter device. VectorNTI 7.1 (Invitrogen) was used for analyzing the resulting sequences.
4. A second copy of each 1:400 dilution of the overnight was made. These second copies were grown in the absence of AHL for 8hrs. This step was to eliminate any accumulated GFP before assaying performance.
5. Samples from both of the cultures were induced with a high input level of AHL (100nM) at 37C with shaking at 70 rpm for 45mins. Single-cell fluorescence measurements were carried out on a Becton-Dickinson FACScan flow cytometer with a 488nm Argon excitation laser and 525nm emission filter. FACScan data were analyzed using Cell Quest and FlowJo. During each flow-cytometer measurement, data was collected from 50000 cells. 2μl of Sphero fluorescent beads (0.87μm ø, Spherotech) in 500μl H₂O were used as a control for temporal variation of cytometer variation.

Old Experimental details

Culture Propagation

1. Pick a colony from the plate and leave for overnight. Dilute 1:400 in the morning.
2. At 8pm Inoculate two cultures from this culture, one with saturating AHL, one with no AHL, [5ml M9+Kan]. Use 50μl of 10μM AHL (this should give 100nM AHL, which is saturating). Its crucially important that they both start from the same, single colonyto make sure there is minimal possibility for mutants existing in the starting culture.
3. Grow the culture overnight @ 37C in the roller.
4. Next day at 10am, measure the OD with the portable spec and then dilute both cultures 1:400 [12.5μl culture, 5ml M9+Kan] and make two copies of each culture. One copy is used for propagation (the "propagation cultures") and the other will be used for testing device function (the "test cultures"), see below.
5. Grow the propagation cultures through the day @ 37C in the roller.
6. After 10 hours (i.e. ~8pm) dilute the propagation cultures by 1:4096. This is based on an assumed 70min doubling time and a 14hr incubation period.
   1. Record the OD of the propagation culture.
   2. Perform the 1:4000 dilution in two steps 1:40 followed by 1:100.
   3. 1:40 - 25μl culture into 975μl M9+Kan.
   4. 1:100 - 50μl of the previous dilution into 5ml M9+Kan +/- AHL.
7. Return these two cultures to the warm room to grow overnight.

Genetic Stability Assay

1. Plate around 100 colonies (there is 10^9 colnies in 1mL. Take 10uL into a 90uL of water this will be 10^7 then dilute 5 times 1:10 and plate the last dilution)
2. Store plates at 4C

Performance Stability Assays

1. During the day, label 16 13mm culture tubes and 16 FACS tubes appropriately for the flow cytometry experiment
2. Add 1.625ml of M9+Kan to each 13mm culture tube.
3. At 6pm, divide each test culture into the eight 13mm culture tubes (375μl per tube) that are preloaded with media. Add 20μl of each AHL concentration.
4. 6:15: Incubate on the flat bed shaker for 45mins.
5. Take 200μl of of each culture and add it to a 96well plate.
6. Place the remainder of the samples on ice.
7. Start a short protocol on the plate reader, no need to add water - this is the assay to test for population-level device function.
8. Run flow cytometry on each of the samples - this is the assay to test for cell-level device function.
9. Place 2μl of Sphero fluorescent beads (0.87μmø) in 500μl H₂O as a control for cytometer variation from day to day.