Evolutionary Stability of Fluorescent Protein Expression

This protocol is a work in progress

This procedure is to monitor the decay of a genetic device that outputs GFP fluorescence as a microbe replicates, it accumulates mutations that lead to a loss in fluorescent signal, and these mutant cells outcompete cells with fully functioning copies of the device.

Procedure: Growth

1. Plate the ancestral strain to isolate single colonies
   • It is very important to start from brightly fluorescent single colonies so that any existing genetic variation in a stock culture is purged (which is very common for devices that lose function rapidly). Only in this case are the observed decay curves and breakage mutations evolutionarily independent!
2. Pick entire single colonies from these plates and resuspend each one in a 10 mL culture in a 50 mL flask. These are your replicate cultures.
   • It is important to get the entire colony, because then one can figure out exactly how many cell divisions have taken place from the single cell that initiated the colony.
3. Each growth cycle (usually 24 h) transfer 100 µL of the overnight culture and into 10 mL of fresh media and either store the culture or immediately take a measurement (methods for this explained below).
   • This 1000-fold dilution equals 10 generations of binary cell division.

Procedure: Measurement

Measurement by Plating

This section is a work in progress

Measurement by Microplate Reader

This section is a work in progress

Measurement by Flow Cytometry

Note

• E. coli fluorescence seems to be relatively stable for up to ~7 days at 4°C in these measurements.

1. Aliquot 100uL of cells into 1.5 mL tubes, add 1 µL of FM 4-64 membrane dye
2. Incubate cells + dye for 10 min, shaking at 37°C.
3. Spin cells down for 5 minutes at 3000 rpm.
4. Remove media and resuspend with an equal volume of saline or PBS.
5. Proceed to using Flow Cytometry to count.
6. After fluorescent has been assayed, plot the %FP over the number of days

Measurement by Next-Gen Sequencing

This section is a work in progress

Expected Results

This section is a work in progress