---+ Co-culture Competition Assays The instructions below include the basic protocol. Be sure to check whether there are variations needed for your specific samples! For example, additional instructions specific for the *Lenski Long-Term Evolution Experiment* are indicated by *LTEE*. ---++ Day –2: Revive Revive the strains to be tested from the freezer by growing one overnight culture of each. Typically, each one is inoculated into 5 ml LB in a test tube. Be sure to include an uninoculated "blank" to test your media stock for contamination. Incubate at 37°C, at 120 RPM for 16 to 24 hours. For _mixed population samples_, you need to revive a representative sample of all the individuals in the population. Inoculate with a volume at least as large as that of a typical transfer during the evolution experiment, taking into account the volume of cryoprotectant added when freezing; 100 µl is often a acceptable volume for mixed population samples. For _clonal isolate samples_, you only need a very small number of cells to start this culture. Either pipette 2 µl of frozen stock culture or use a pipette tip to scape a very small amount of the stock culture from the surface while it is still frozen. *LTEE:* A representative _mixed population sample_ from a DM25 freezer stock is 120 µl (100 µl transfer + 20 µl 80% glycerol used in freezing). *Variations:* For some competitions, it may be more appropriate to revive in DM1000 or another growth 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. (For example, if you are doing a six-fold replication of a competition between strain #1 and strain #2, you should have 6 tubes of strain #1 and 6 tubes of strain #2.) Typically, you will want to inoculate this culture with a sufficient dilution that it's initial density is at least 100-fold below the final 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 the 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 10 ml DM25. Vortex to thoroughly mix. Immediately transfer 100 µl to a dilution tube with 10 ml of saline (10,000&times; dilution). Plate 80 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. Preserving the dilution tubes may prove helpful if the plates do not grow properly. Dilution tubes can be safely stored at 4°C for 24 hours without affecting CFUs for most experiments with _E. coli_. For the LTEE, only 50-60 µl of the ancestor and early clones need to be plated to get a sufficient number of cells. The cell density is reduced later in evolved strains, which is when it is appropriate to plate 80-100 µl. *Variations:* You may need to plate larger or smaller volumes and/or dilutions to get an appropriate number of cells on each plate for counting. The volume you plate should always be int he range 10-100 µl. If you plate only 10-20 µl, you may want to add 50 µl of saline to the plate before spreading. ---++ 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 80 µl of this second dilution on a TA plate. Grow plates for 16-24 hrs at 37°C before counting. Preserving the dilution tubes may prove helpful if the plates do not grow properly. Dilution tubes can be safely stored at 4°C. For this day, only the second dilution tube (the one from which the samples were actually plated) should be stored. ---++ 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 = log(M_A) / log(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 tested. In these cases it is better to use selection rates (r) to measure fitness as discussed [[https://lenski.mmg.msu.edu/ecoli/srvsrf.html][here]]. ---++ Expected Results *LTEE:* With 12 replicates and a three day competition, the relative fitness of two closely matched strains should be measurable with a precision of ± 1% (95% confidence intervals). ---++ References _This protocol and discussion are modified from the web pages of [[https://lenski.mmg.msu.edu][Richard Lenski]]_.

   

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Contributors to this topic JeffreyBarrick, AurkoDasgupta, LindseyWolf, CraigBarnhart

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