Competition Assays for Evolvability Lines

Serial transfer of 3.5 µl of culture to 900 µl of fresh medium achieves 8.0120 generations per day (assuming the 3.5 µl is transfered with the pin tool and it also takes 3.5 µl away after the transfer). This is a 258.1x dilution.

Generally DM0 + 0.01% w/v of a single carbon compound is used as the growth medium. For glucose, this allows growth to approximately 2 x 10⁸ cells per ml. Other compounds may not support this level of growth.

Materials

• 96-well pin tool that transfers 3.5 µl.
• For each competition plate:
  • 5 deep 96-well plates filled with 900 µl per well of growth medium.
  • 1 deep 96-well plate filled with 900 µl per well of saline.
  • 2 deep 96-well plates filled with 600 µl per well of saline.
• For each competition replicate:
  • 2 TA plates.

Procedure

Day -2: Reviving Frozen Stocks

Revive the strains to be tested from the freezer by growing them in deep 96-well plates containing 900 ml of the competition medium per well. If using a mixed population, be sure to use at least 3.5 µl of inoculate from the frozen stock to ensure that the sample is representative of the population.

You will need two plates for each competiton plate that you are creating, one for reviving each of the strains to be competed separately (for example, one for the evolved strains and one for the ancestor strains). Frozen plates of 606/607 in a checkerboard pattern are available to start the ancestor strains for competitions.

Grow 24 hours at 37°C with shaking at 120 rpm.

Day -1: Preconditioning Cultures

Transfer 3.5 µl from each well to a fresh deep 96-well plate filled with 900 µl of the growth medium per well. After this second cycle of dilution and growth, cells have presumably reached a physiological steady state similar to what occurs during the evolution experiment and an accurate estimate of their fitness under those conditions can be made.

Grow exactly 24 hours at 37°C with shaking at 120 rpm.

Day 0: Begin Competition

Pair up plates containing strains that will be competed. Mix these by slowly circulating the pin tool before using a multichannel pipetteman to transfer 1.75 µl from each of the two plates into a new plate with 900 µl growth medium per well.

Adding 1/2 the normal transfer volume of each strain is necessary since we want the total cell number of both strains we are competing to be approximately at what it would be during a normal day of growth, i.e. we need to achieve 8 generations under the exact conditions of evolution.

Immediately make a dilution of the competition plates by transferring 3.5 µl from each well into a plate containing 900 µl of saline per well.

Move competition microplates to the incubator. Grow exactly 24 hours at 37°C with shaking at 120 rpm.

Plate 5-200 µl of the dilution on TA plates. A volume should be chosen that yields 100-300 cells. Use the following table as a guide for the densities that the REL606/607 ancestors achieve after growth in each of the carbon compounds that was tested. The densities of evolved strains on a given carbon compound may have changed. It may need to be empirically determined before performing the competition. (If the final density changes by more than about 2-fold, then some of the assumptions of this method for determining fitness are challenged. The amount of ancestor cells in the initial mixture could be adjusted to compensate, but there may still be frequency dependent effects on fitness.)

Cell Density	Volume to Plate	Carbon Compounds
4 x 108	20 µl	1, 2, 17
2 x 108	40 µl	3, 4, 6, 7, 8, 9, 10, 18, 19
1 x 108	80 µl	5, 13, 14, 15, 22, 24
5 x 107	160 µl	12, 16, 21
2.5 x 107	320 µl	11, 20, 23

This is to achieve ~180 cells per plate. The final dilution factor is 4.4 x 10⁴.

Grow plates for 16-24 hours at 37°C.

Day +1: Count Initial Plates and End Competition

Count the numbers of red and white colonies on each TA plate to determine the initial frequencies of the two strains in the competition before growth.

Transfer 3.5 µl from each well of the competition plate into a new deep 96-well plate containing 900 µl of saline per well. As was done on Day 5, make one further serial dilution of 3.5 µl per well into a plate with 600 µl of saline per well and plate an appropriate volume of this final dilution on TA plates. (This will generally be the same volume plated on Day 0. However, it can be adjusted to give a more countable )

Day +2: Count Final Plates

Count the numbers of red and white colonies on each TA plate to determine the final frequencies of the two strains in the competition after growth. Calculate the relative fitness of the two strains.

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 = log [N_A(f) / N_A(i)] = log [PC_A(f) * DF / PC_A(i)]

M_B = log [N_B(f)] / N_B(i)] = log [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.

Variations

For measuring fitness values more precisely, you can continue to dilute and grow under the same conditions 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. Beware that evolution can happen during longer competitions depending on how strong the selective pressures are.

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