---+ Co-culture Competition Assays Instructions for the *LTEE* are specific for the Lenski Long-Term Evolution Experiment with _E. coli_. ---++ Day –2: Revive Revive the strains to be tested from the freezer by growing them in one flask or test tube each in LB. Be sure to include an uninoculated blank to test your LB stock for contamination. If using a mixed population, be sure to use a volume at least as large as that of a typical transfer to ensure that the sample is representative. If using clones, scrape a small amount from a frozen freezer stock to inoculate. Incubate at 37°C, at 120 RPM for 16 to 24 hours. LTEE: Inoculate with 100 µl of frozen mixed population stocks, which is approximately the daily transfer dilution. 10 ml of LB in a 50 ml flask or 3-5 ml of LB in a test tube may be used. Variations: For some competitions, it may be more appropriate to revive in a medium other than LB. ---++ Day –1: Precondition Prepare one flask, test tube, or well for each time a strain will be used in a co-culture competition replicate. Typically, you will want to inoculate this culture with at least a 100-fold dilution of the cell density it can support after growth, so that it goes through several doublings and the media from the first day is diluted sufficiently by new media. Incubate at 37°C, at 120 RPM for 24 hours. LTEE: Transfer 100 µl of overnight LB culture into a dilution tube with 10 ml of saline (100&times; dilution). Then transfer 100 µl of this into 10 ml of DM25 (10,000&times; total dilution). The extra dilution is necessary because DM25 media supports only ~1/100th the final density of LB. ---++ Day 0: Begin Competition 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. Return mixed strains to incubator and incubate at 37°C, 120 RPM for 24 hours. LTEE: Transfer 50 µl of each of the strains to be competed to a new 50 ml flask containing DM25. Immediately transfer 100 µl to a dilution tube with 10 ml of saline (10,000&times; dilution). Plate 50 µl of this dilution on a TA plate. Grow cultures for 24 hrs at 37°C shaking at 120 rpm. Grow plates for 16-24 hrs at 37°C before counting. ---++ Day 1: Finish Competition 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). LTEE: Transfer 100 µl of each competition to a dilution tube with 10 ml of saline (100&times; dilution). Then transfer 100 µl of this dilution to a second dilution tube with 10 ml of saline (10,000&times; total dilution). Plate 50 µl of this second dilution on a TA plate. Grow plates for 16-24 hrs at 37°C before counting. ---++ 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_A and M_B) 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_A = N_A(f) / N_A(i) = PC_A(f) * DF / PC_A(i) M_B = N_B(f) / N_B(i) = PC_B(f) * DF / PC_B(i) W = M_A / M_B 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]]_

   

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