(r1) Barrick Lab :: ProtocolsFluorescentProteinEvolutionaryStability

---+ Evolutionary Stability of Fluorescent Protein Expression

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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
      * %ICON{warning}% 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!
   1 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.
   1 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

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---+++ Measurement by Microplate Reader

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---+++ 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 [[https://www.thermofisher.com/order/catalog/product/T13320][FM 4-64 membrane dye]]
   1 Incubate cells + dye for 10 min, shaking at 37°C.
   1 Spin cells down for 5 minutes at 3000 rpm.
   1 Remove media and resuspend with an equal volume of saline or PBS.
   1 Proceed to using Flow Cytometry to count.
   1 After fluorescent has been assayed, plot the %FP over the number of days 

---+++ Measurement by Next-Gen Sequencing

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---++ Expected Results

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Barrick Lab > ProtocolList > ProtocolsFluorescentProteinEvolutionaryStability

Contributors to this topic

JeffreyBarrick, LucyLeblanc, DennisMishler, DaciaLeon

Topic revision: r1 - 2016-02-23 - 04:00:48 - Main.JeffreyBarrick