---+++ Competition Assays for Evolvability Lines ---++++ Day -2 Revive the strains to be tested from the freezer by growing them in 1 ml of LB in deep 96-well plates. If using a mixed population, be sure to use at least 3.5 µl of innoculate from the frozen stock to ensure that the sample is representative of the population. Grow 16-24 hours. ---++++ Day -1 Prepare a deep 96-well plate with 900 µl of saline in each well using the multichannel repeat pipettor. Transfer 100 µl from the overnight in LB to this plate with a multichannel pipettemen. Mix by pipetting up and down several times. This makes a 10-fold dilution that has a cell density of approximately 5 x 10<sup>8</sup> cells/ml, which is approximately the saturating cell density during these long term evolvability experiments. Transfer 3.5 µl from this dilution plate to a new plate filled with 1 ml per well of the medium to be used in the competition using a 96-well pin tool or a multichannel pipettor. This achieves approximately the density experienced by cells after a transfer. Grow exactly 24 hours. ---++++ Day 0 Mix together 200-fold dilutions of the two strains to be competed in the medium to be tested (giving a 100-fold overall dilution in cell number). Immediately make a dilution that will yield 100-500 cells, and plate on TA. These counts give the initial frequencies of the two strains in the competition. ---++++ Day 1 After exactly 24 hours. Plate a dilution of each culture on TA (typically this will be 100-fold more than the amount plated on Day 0). ---++++ Variations For measuring fitness values more precisely, you can continue to serially dilute for multiple days before plating. This is useful, for example, when showing that the mutation in an Ara+ revertant of an Ara- REL606-based strain is neutral. But beware that evolution can happen during this longer time-period depending on how strong the selective pressures are. ---+++ Calculating Relative Fitness (W) The relative fitness (W) of strains A relative to straind B is the ratio of their Malthusian parameters (M<sub>A</sub> and M<sub>B</sub>) over the course of a representative growth cycle. N = cell number. <br> PC = plate count on TA. <br> DF = dilution factor of all transfers combined.<br> i and f are the initial and final time points. M<sub>A</sub> = N<sub>A</sub>(f) / N<sub>A</sub>(i) = PC<sub>A</sub>(f) * DF / PC<sub>A</sub>(i) M<sub>B</sub> = N<sub>B</sub>(f) / N<sub>B</sub>(i) = PC<sub>B</sub>(f) * DF / PC<sub>B</sub>(i) W = M<sub>A</sub> / M<sub>B</sub> Note, that there are problems with this measurement under conditions where: (1) One or both populations are declining in numbers over the course of the competition -- which would lead to negative W values -- or (2) There is a large difference in fitness between the two strains being tests. In these cases it is better to use selection rates (r) to measure fitness as discussed [[http://myxo.css.msu.edu/ecoli/srvsrf.html][here]]. _This protocol and discussion are modified from the web pages of [[http://www.msu.edu/~lenski/][Richard Lenski]]_ -- Main.JeffreyBarrick - 17 Sep 2007
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Topic revision: r1 - 2007-09-18 - JeffreyBarrick