(r2) 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

1) Plan out which wells will contain what sample. You can print out one of the 96 well plate templates available [[http://www.cellsignet.com/media/plates/96.jpg][online]]. Generally, each condition should be done in quadruplicate (or triplicate depending on space concerns). <br>
2) After vortexing your sample, aliquot 100&#956;L at a time into each well. You should use the appropriate type of plate for your application: black walled, clear bottom for fluorescence (white walled, clear bottom is generally for luminescence and completely clear is generally for other applications, like measuring OD600). <br>
3) Ensure that you have adequate controls. At the very least, have a nonfluorescent negative control of E. coli in the same media grown under the same conditions, either empty vector or isolated from a colony that had lost its fluorescence. <br>
4) Turn on the plate reader - there is a power button in the back next to the power cord - and then turn on the Magellan software on the computer next to it. Not doing it in this order may cause the software to fail to recognize the machine. <br>
5) Select the option that says "raw data" and eventually you will find yourself at a configuration screen. <br>
6) First, select the brand and type of your plate next to the "Plate definition" dropdown menu. <br>
7) Second, using ctrl click, select the wells you want to measure. <br>
8) Third, you must add a measurement step. Double click "Fluorescence Intensity." Set your Excitation and Emission values to integers appropriate for your fluorophore and change Gain to "Optimal". You can also manually set gain by testing different values: generally, a higher gain detects smaller amounts of fluorescence; if you get overflow reads in several wells, you should set the gain lower. <br>
9) Fourth, perform the measurements and save your data. Unfortunately, there is no Excel installed on that computer, so "Copy to Excel" is not a possibility at this point. Data is saved by default as ".wsp" files. (Still looking for a workaround - there is also a "Copy as ASCII" option that I haven't tested yet). <br>
10) Finally, after performing this over a number of days, calculate how RFU changes as a function of time. <p>

Troubleshooting: <br>
a) If you are not sure what the excitation/emission values for your fluorophore are, here is a handy reference courtesy of Simon: [[https://slack-files.com/files-pri-safe/T04QHM990-F0Q59MJMD/filter_combinations_fps_spectra.pdf?c=1457108292-b10ad8f9e80d418c5d5e0ca0a9b5d994bb51795d][Spectra of Common Fluorophores]]. <br>
b) You can BLAST the gene sequence of your fluorophore to check which it matches (most likely a variant of EGFP). If you are still not sure or getting bizarre results, you may want to do a "Fluorescence Intensity Scan" where you set the plate reader to test numerous wavelengths of excitation in a certain range and then look at the spectra to see where the peaks (highest RFUs) are, in terms of wavelength. <br>

---+++ 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: r2 - 2016-03-04 - 16:18:51 - Main.LucyLeblanc