Revive and Initiate Populations

1. Revive the E. coli strain with the genetic device plasmid from a freezer stock stored at –80°C by inoculating 5mL LB+Amp in a test tube and growing at 37°C with orbital shaking.

2. Plate a dilution on selective LB+Amp agar that will yield 50-150 well-separated colonies.

3. Incubate plate for 24 hours at 37°C.

4. Fill 6-12 separate test tubes with 5mL LB+Amp. Inoculate each test tube with all of the cells from a different colony on the agar plate.

CRITICALOnly pick colonies that are fully fluorescent. Colonies that are not fluorescent are derived from cells that have already lost device function!

CRITICALYou must transfer all of the cells in the chosen colony to the test tube. This can be accomplished by digging a divot out of the agar around an isolated colony with a pipette tip and transferring both the agar and the intact colony on top of it into the test tube.

Propagate Populations and Monitor Device Function

5. Grow test tube cultures for 24 hours at 37°C with orbital shaking. They should reach a saturating cell density in this amount of time.

6. Measure the fluorescence of each culture by one of these methods:

A. Plating a dilution on LB+Amp agar and counting the percentage of colonies that remain fully fluorescent (% GFP+ cells) after 24 hours of growth at 37°C.

B. Using a microplate reader to measure the GFP signal normalized to optical density in a microplate reader (GFP signal).

C. Using a flow cytometer to count the percentage of fully fluorescent cells (% GFP+ cells).

7. Transfer 5µL of culture from each test tube to 5mL of fresh LB+Amp in a new test tube.

8. Return to step 6. Repeat until GFP expression is reduced or completely lost.

Data Analysis

9. Graph the function of the device (as remaining % GFP+ cells or remaining % GFP signal) versus time measured in generations (binary cell divisions). The number of generations that have elapsed is calculated by assuming each population began as a single cell. Therefore the number of generations is equal to the log base 2 of the product of the final cell number in a test tube that has growth to saturation times the cumulative dilution factor up to that point. In this cane, the dilution factor is 1000x times the number of days of propagation, in this case.

To form each initial colony from a single cell requires ~25 generations of binary cell division. Several more generations are required for these cells to grow to a saturating culture in a test tube. Then, each further growth cycle after a 1000x dilution adds another ~10 generations.

10. Examine the graph to find when the earliest time when the average function of the device across the six replicate populations drops below 50%. This is the evolutionary half-life of this genetic device. More stable devices have longer evolutionary half-lives.