Difference: ProceduresEvolvabilityCompetitions (1 vs. 11)

Revision 112008-05-22 - JeffreyBarrick

 
META TOPICPARENT name="WebHome"

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 (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.

Changed:
<
<
MA = NA(f) / NA(i) = PCA(f) * DF / PCA(i)
>
>
MA = log [NA(f) / NA(i)] = log [PCA(f) * DF / PCA(i)]
 
Changed:
<
<
MB = NB(f) / NB(i) = PCB(f) * DF / PCB(i)
>
>
MB = log [NB(f)] / NB(i)] = log [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.

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

Revision 102008-01-16 - JeffreyBarrick

 
META TOPICPARENT name="WebHome"

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

Changed:
<
<
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.
>
>
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.
 
Changed:
<
<
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.
>
>
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.
 
Changed:
<
<
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.
>
>
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 (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.

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

Revision 92007-11-10 - JeffreyBarrick

 
META TOPICPARENT name="WebHome"

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

Changed:
<
<

Day 1: Reviving Frozen Stocks

>
>

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.
Changed:
<
<
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.
>
>
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.
 
Changed:
<
<
Grow 40-48 hours at 37°C with shaking at 120 rpm.
>
>
Grow 24 hours at 37°C with shaking at 120 rpm.
 
Changed:
<
<

Day 3: Preconditioning Cultures

>
>

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.
Changed:
<
<
Grow exactly 48 hours at 37°C with shaking at 120 rpm.
>
>
Grow exactly 24 hours at 37°C with shaking at 120 rpm.
 
Changed:
<
<

Day 5: Begin Competition

>
>

Day 0: Begin Competition

  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.

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.

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.

Changed:
<
<
Move competition microplates to the incubator. Grow exactly 48 hours at 37°C with shaking at 120 rpm.
>
>
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.

Changed:
<
<

Day 6: Count Initial Plates

>
>

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.
Changed:
<
<

Day 7: End Competition

>
>
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 )
 
Changed:
<
<
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 be the same volume plated on Day 5.)
>
>

Day +2: Count Final Plates

Deleted:
<
<

Day 8: 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 (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.

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

Revision 82007-10-30 - JeffreyBarrick

 
META TOPICPARENT name="WebHome"

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.
Changed:
<
<
    • 4 deep 96-well plates filled with 600 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 1: 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 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.

Grow 40-48 hours at 37°C with shaking at 120 rpm.

Day 3: 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 48 hours at 37°C with shaking at 120 rpm.

Day 5: Begin Competition

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.

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.

Changed:
<
<
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.
>
>
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.
  Move competition microplates to the incubator. Grow exactly 48 hours at 37°C with shaking at 120 rpm.
Changed:
<
<
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.)
>
>
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
Changed:
<
<
4 x 108 12.5 µl 1, 2, 17
2 x 108 25 µl 3, 4, 6, 7, 8, 9, 10, 18, 19
1 x 108 50 µl 5, 13, 14, 15, 22, 24
5 x 107 100 µl 12, 16, 21
2.5 x 107 200 µl 11, 20, 23
This is to achieve ~200 cells per plate. The final dilution factor is 7.56 x 106.
>
>
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 6: Count Initial Plates

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

Changed:
<
<
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.)
>
>
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 be the same volume plated on Day 5.)
 

Day 8: 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 (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.

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

Revision 72007-10-19 - JeffreyBarrick

 
META TOPICPARENT name="WebHome"

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.
Changed:
<
<
    • 4 deep 96-well plate filled with 600 µl per well of saline.
>
>
    • 4 deep 96-well plates filled with 600 µl per well of saline.
 
  • For each competition replicate:
    • 2 TA plates.
Added:
>
>

Procedure

 

Day 1: 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 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.

Grow 40-48 hours at 37°C with shaking at 120 rpm.

Day 3: 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 48 hours at 37°C with shaking at 120 rpm.

Day 5: Begin Competition

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.

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.

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.

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.)

Cell Density Volume to Plate Carbon Compounds
Changed:
<
<
4 x 108 6.25 µl  
2 x 108 12.5 µl  
1 x 108 25 µl  
5 x 107 50 µl  
2.5 x 107 100 µl  
This is to achieve ~100 cells per plate. The final dilution factor is 7.56 x 106.
>
>
4 x 108 12.5 µl 1, 2, 17
2 x 108 25 µl 3, 4, 6, 7, 8, 9, 10, 18, 19
1 x 108 50 µl 5, 13, 14, 15, 22, 24
5 x 107 100 µl 12, 16, 21
2.5 x 107 200 µl 11, 20, 23
This is to achieve ~200 cells per plate. The final dilution factor is 7.56 x 106.
  Grow plates for 16-24 hours at 37°C.

Day 6: Count Initial Plates

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

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.)

Day 8: 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 (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.

Changed:
<
<

Variations

>
>

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

Revision 62007-10-19 - JeffreyBarrick

 
META TOPICPARENT name="WebHome"

Competition Assays for Evolvability Lines

Changed:
<
<
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.
 
Changed:
<
<
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.
 
Deleted:
<
<
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

Changed:
<
<
  • 96-Well Pin Tool that transfers 3.5 µl.
>
>
  • 96-well pin tool that transfers 3.5 µl.
Added:
>
>
  • 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.
    • 4 deep 96-well plate filled with 600 µl per well of saline.
  • For each competition replicate:
    • 2 TA plates.
 

Day 1: Reviving Frozen Stocks

Deleted:
<
<
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.
 
Added:
>
>
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.
Changed:
<
<
Grow 40-48 hours.
>
>
Grow 40-48 hours at 37°C with shaking at 120 rpm.
 

Day 3: Preconditioning Cultures

Deleted:
<
<
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.
 
Changed:
<
<
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.
 
Added:
>
>
Grow exactly 48 hours at 37°C with shaking at 120 rpm.
 

Day 5: Begin Competition

Changed:
<
<
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.
 
Changed:
<
<
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.
 
Changed:
<
<
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.
 
Added:
>
>
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.)

Cell Density Volume to Plate Carbon Compounds
4 x 108 6.25 µl  
2 x 108 12.5 µl  
1 x 108 25 µl  
5 x 107 50 µl  
2.5 x 107 100 µl  
This is to achieve ~100 cells per plate. The final dilution factor is 7.56 x 106.

Grow plates for 16-24 hours at 37°C.

 

Day 6: Count Initial Plates

Added:
>
>
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

Changed:
<
<
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.)
 
Deleted:
<
<
Count the plates from Day 0.
 

Day 8: Count Final Plates

Changed:
<
<
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.
 
Deleted:
<
<

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. 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.

Changed:
<
<
This protocol and discussion are modified from the web pages of Richard Lenski
>
>

Variations

Added:
>
>
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.
 
Changed:
<
<
>
>
This protocol and discussion are adapted from the web pages of Richard Lenski
Deleted:
<
<

-- Main.JeffreyBarrick - 17 Sep 2007

Revision 52007-10-19 - JeffreyBarrick

 
META TOPICPARENT name="WebHome"

Competition Assays for Evolvability Lines

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 258.1 x dilution.

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

  • 96-Well Pin Tool that transfers 3.5 µl.
Changed:
<
<

Day -2: Reviving Frozen Stocks

Revive the strains to be tested from the freezer by growing them in 1 ml of LB 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. Grow 16-24 hours.
>
>

Day 1: Reviving Frozen Stocks

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.
 
Changed:
<
<

Day -1: Preconditioning Cultures

>
>
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.
Added:
>
>
Grow 40-48 hours.

Day 3: Preconditioning Cultures

 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.

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.

Changed:
<
<

Day 0: Begin Competition

>
>

Day 5: Begin Competition

  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.)

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.

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.

Changed:
<
<

Day 1: End Competition / Count Initial Plates

>
>

Day 6: Count Initial Plates

 
Added:
>
>

Day 7: End Competition

 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.

Count the plates from Day 0.

Changed:
<
<

Day 2: Count Final Plates

>
>

Day 8: Count Final Plates

  Count the plates

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. 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.

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

-- Main.JeffreyBarrick - 17 Sep 2007

Revision 42007-09-18 - JeffreyBarrick

 
META TOPICPARENT name="WebHome"

Competition Assays for Evolvability Lines

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 258.1 x dilution.

Changed:
<
<
0.01% of the carbon compound is used. For glucose, this allows growth to approximately 2 x 107 cells per ml. Other compounds may not support this level of growth.
>
>
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

  • 96-Well Pin Tool that transfers 3.5 µl.

Day -2: Reviving Frozen Stocks

Changed:
<
<
Revive the strains to be tested from the freezer by growing them in 1 ml of LB in deep 96-well plates. If using a mixed population, be sure to use at least 3.5 µl of innoculate from the frozen stock to ensure that the sample is representative of the population. Grow 16-24 hours.
>
>
Revive the strains to be tested from the freezer by growing them in 1 ml of LB 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. Grow 16-24 hours.
 

Day -1: Preconditioning Cultures

Changed:
<
<
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 107 cells/ml, which is roughly the saturating cell density achieved during these long term evolvability experiments.
>
>
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.
  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.

Day 0: Begin Competition

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.)

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.

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.

Day 1: End Competition / Count Initial Plates

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.

Count the plates from Day 0.

Day 2: Count Final Plates

Count the plates

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. 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.

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

-- Main.JeffreyBarrick - 17 Sep 2007

Revision 32007-09-18 - JeffreyBarrick

 
META TOPICPARENT name="WebHome"

Competition Assays for Evolvability Lines

Deleted:
<
<
5E9 5E8

2E8

5E7 2E7 1E6

 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 258.1 x dilution.

Added:
>
>
0.01% of the carbon compound is used. For glucose, this allows growth to approximately 2 x 107 cells per ml. Other compounds may not support this level of growth.
 

Materials

  • 96-Well Pin Tool that transfers 3.5 µl.

Day -2: Reviving Frozen Stocks

Revive the strains to be tested from the freezer by growing them in 1 ml of LB in deep 96-well plates. If using a mixed population, be sure to use at least 3.5 µl of innoculate from the frozen stock to ensure that the sample is representative of the population. Grow 16-24 hours.

Day -1: Preconditioning Cultures

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 107 cells/ml, which is roughly the saturating cell density achieved during these long term evolvability experiments.

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.

Day 0: Begin Competition

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.)

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.

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.

Day 1: End Competition / Count Initial Plates

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.

Count the plates from Day 0.

Day 2: Count Final Plates

Count the plates

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. 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.

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

-- Main.JeffreyBarrick - 17 Sep 2007

Revision 22007-09-18 - JeffreyBarrick

 
META TOPICPARENT name="WebHome"

Competition Assays for Evolvability Lines

Changed:
<
<

Day -2

>
>
5E9
Added:
>
>
5E8

2E8

5E7 2E7 1E6

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 258.1 x dilution.

Materials

  • 96-Well Pin Tool that transfers 3.5 µl.

Day -2: Reviving Frozen Stocks

 Revive the strains to be tested from the freezer by growing them in 1 ml of LB in deep 96-well plates. If using a mixed population, be sure to use at least 3.5 µl of innoculate from the frozen stock to ensure that the sample is representative of the population. Grow 16-24 hours.
Changed:
<
<

Day -1

Prepare a deep 96-well plate with 900 µl of saline in each well using the multichannel repeat pipettor. Transfer 100 µl from the overnight in LB to this plate with a multichannel pipettemen. Mix by pipetting up and down several times. This makes a 10-fold dilution that has a cell density of approximately 5 x 108 cells/ml, which is approximately the saturating cell density during these long term evolvability experiments.
>
>

Day -1: Preconditioning Cultures

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 107 cells/ml, which is roughly the saturating cell density achieved during these long term evolvability experiments.
 
Changed:
<
<
Transfer 3.5 µl from this dilution plate to a new plate filled with 1 ml per well of the medium to be used in the competition using a 96-well pin tool or a multichannel pipettor. This achieves approximately the density experienced by cells after a transfer. Grow exactly 24 hours.
>
>
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.
 
Changed:
<
<

Day 0

>
>

Day 0: Begin Competition

Deleted:
<
<
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.
 
Changed:
<
<

Day 1

>
>
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.)
Deleted:
<
<
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).
 
Added:
>
>
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.

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.

Day 1: End Competition / Count Initial Plates

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.

Count the plates from Day 0.

Day 2: Count Final Plates

Count the plates

 

Variations

Changed:
<
<
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.
>
>
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. 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.

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

-- Main.JeffreyBarrick - 17 Sep 2007

Revision 12007-09-18 - JeffreyBarrick

 
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Competition Assays for Evolvability Lines

Day -2

Revive the strains to be tested from the freezer by growing them in 1 ml of LB in deep 96-well plates. If using a mixed population, be sure to use at least 3.5 µl of innoculate from the frozen stock to ensure that the sample is representative of the population. Grow 16-24 hours.

Day -1

Prepare a deep 96-well plate with 900 µl of saline in each well using the multichannel repeat pipettor. Transfer 100 µl from the overnight in LB to this plate with a multichannel pipettemen. Mix by pipetting up and down several times. This makes a 10-fold dilution that has a cell density of approximately 5 x 108 cells/ml, which is approximately the saturating cell density during these long term evolvability experiments.

Transfer 3.5 µl from this dilution plate to a new plate filled with 1 ml per well of the medium to be used in the competition using a 96-well pin tool or a multichannel pipettor. This achieves approximately the density experienced by cells after a transfer. Grow exactly 24 hours.

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 (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.

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

-- Main.JeffreyBarrick - 17 Sep 2007

 
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