Difference: ProceduresEvolvabilityCompetitions (5 vs. 6)
Revision 62007-10-19 - JeffreyBarrick
Competition Assays for Evolvability Lines | |||||||||||||||||||
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| < < | The 96-well pin tool transfers 3.5 µl. | ||||||||||||||||||
| > > | 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. | ||||||||||||||||||
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| < < | 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 258.1 x 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. | ||||||||||||||||||
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| < < | 0.01% of the carbon compound is used. For glucose, this allows growth to approximately 2 x 108 cells per ml. Other compounds may not support this level of growth. | ||||||||||||||||||
Materials | |||||||||||||||||||
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Day 1: Reviving Frozen Stocks | |||||||||||||||||||
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| < < | Revive the strains to be tested from the freezer by growing them in 900 ml of the competition medium in deep 96-well plates. 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. | ||||||||||||||||||
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| > > | 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 competitoon 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. | |||||||||||||||||||
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| < < | Grow 40-48 hours. | ||||||||||||||||||
| > > | Grow 40-48 hours at 37°C with shaking at 120 rpm. | ||||||||||||||||||
Day 3: Preconditioning Cultures | |||||||||||||||||||
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| < < | Prepare a deep 96-well plate with 100 µl of saline in each well using the multichannel repeat pipettor. Transfer 3.5 µl from the overnight in LB to this plate with a 96-well pin tool or multichannel pipettemen. Mix by stirring with the pins or pipetting up and down several times. This makes a 25-fold dilution that has a cell density of approximately 2 x 108 cells/ml, which is roughly the saturating cell density achieved during these long term evolvability experiments. | ||||||||||||||||||
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| < < | Transfer 3.5 µl from this dilution plate to a new plate filled with 900 µl per well of the medium to be used in the competition using a 96-well pin tool or multichannel pipettor. This achieves approximately the density experienced by cells after a transfer. Grow exactly 24 hours. | ||||||||||||||||||
| > > | 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. | ||||||||||||||||||
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| > > | Grow exactly 48 hours at 37°C with shaking at 120 rpm. | ||||||||||||||||||
Day 5: Begin Competition | |||||||||||||||||||
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| < < | Add 900 µl of DM0 to each well and mix to achieve a 2-fold dilution. (This 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.) | ||||||||||||||||||
| > > | Add 900 µl of DM0 to every well in each of these plates. Pair up plates containing strains that will be competed. Mix these by slowly circulating the pin tool before transferring 3.5 µl from each of the two plates into a new plate with 900 µl growth medium per well. | ||||||||||||||||||
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| < < | Transfer 3.5 µl of each of the two strains to be competed to 900 µl of the medium to be used in the competition. Immediately make a dilution by taking 3.5 µl of each competitions into 900 µl of saline. Put the competition in the incubator at 37°C. | ||||||||||||||||||
| > > | Creating the 2-fold dilutions of each strain before mixing them 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. | ||||||||||||||||||
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| < < | Plate 100 µl of the dilution on TA plates. This dilution should yield 100-500 cells. These counts give the initial frequencies of the two strains in the competition. | ||||||||||||||||||
| > > | Immediately make a dilution of the competition plates by transferring 3.5 µl from each well into a plate containing 600 µl of saline per well. Dilute 3.5 µl from each well of this first serial dilution plate into a second plate containing 600 µl of saline per well. | ||||||||||||||||||
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| > > | Move competition microplates to the incubator. Grow exactly 48 hours at 37°C with shaking at 120 rpm.
Plate 5-200 µl of the final dilution on TA plates. A dilution should be chosen to yield 100-300 cells. Use the following table as a guide for the densities that the ancestor achieves 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 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.)
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Day 6: Count Initial Plates | |||||||||||||||||||
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| > > | 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. | ||||||||||||||||||
Day 7: End Competition | |||||||||||||||||||
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| < < | After exactly 24 hours of growth. Transfer 3.5 µl of each competitions into 900 µl of saline twice.Plate 100 µl of the dilution on TA plates. This dilution should yield 100-500 cells. These counts give the initial frequencies of the two strains in the competition. | ||||||||||||||||||
| > > | Transfer 3.5 µl from each well of the competition plate into a new plate containing 900 µl of saline per well. As was done on Day 5, make two further serial dilutions of 3.5 µl per well into plates with 600 µl of saline per well and plate an appropriate volume of this final dilution on TA plates. (This will be the same volume plated on Day 5.) | ||||||||||||||||||
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| < < | Count the plates from Day 0. | ||||||||||||||||||
Day 8: Count Final Plates | |||||||||||||||||||
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| < < | Count the 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. | ||||||||||||||||||
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| < < | VariationsFor 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. Beware that evolution can happen during longer competitions 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 (MA and MB) 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. MA = NA(f) / NA(i) = PCA(f) * DF / PCA(i) MB = NB(f) / NB(i) = PCB(f) * DF / PCB(i) W = MA / MB 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. | |||||||||||||||||||
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| < < | This protocol and discussion are modified from the web pages of Richard Lenski | ||||||||||||||||||
| > > | Variations | ||||||||||||||||||
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| > > | 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. | ||||||||||||||||||
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| > > | This protocol and discussion are adapted from the web pages of Richard Lenski | ||||||||||||||||||
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| < < | -- Main.JeffreyBarrick - 17 Sep 2007 | ||||||||||||||||||
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