Competition Assays for the Lenski Long Term E. coli Lines

Day -2

Revive the strains to be tested from the freezer by growing them in one flask or test tube each in LB. 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.

Day -1

Prepare one flask, test tube, or well in the medium to be tested for each replicate use of each strain. Typically, you will want to inoculate this culture with a 100-fold dilution of the cell density it can support from the LB overnight.

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_A and M_B) over the course of a representative growth cycle.

N = cell number.
PC = plate count on TA.
DF = dilution factor of all transfers combined.
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 here.

This protocol and discussion are modified from the web pages of Richard Lenski