Jul 14, 2023

Public workspaceLTEE Media Recipes V.1

DOI
https://dx.doi.org/10.17504/protocols.io.n92ldpy5ol5b/v1
  • Jesus Chavarria-Palma1,
  • Jeffrey Barrick2
  • 1University of Texas at Austin;
  • 2The University of Texas at Austin
  • the-ltee
Jeffrey E Barrick
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DOI: https://dx.doi.org/10.17504/protocols.io.n92ldpy5ol5b/v1
Protocol Citation: Jesus Chavarria-Palma, Jeffrey Barrick 2023. LTEE Media Recipes. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldpy5ol5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 27, 2022
Last Modified: July 14, 2023
Protocol Integer ID: 70511
Keywords: ltee media recipe, tetrazolium arabinose, liquid medium, growth media, glucose, sugar concentration, cells per ml, solidifying agent, standard minimal medium, same basic composition as for dm, phase density
Funders Acknowledgements:
National Science Foundation
Grant ID: DEB-1951307
Abstract
Growth media used by the long-term E. coli evolution experiment.
  • TA: Tetrazolium Arabinose for distinguishing Ara- and Ara+ strains in most competition assays. Colonies of Ara- strains typically appear red on TA agar, while those of Ara+ strains appear white. TA plates are generally incubated at 37°C for 24 h.
  • DM: The basic medium used for propagating the long-term lines is Davis Minimal broth supplemented with glucose at a concentration of 25 mg per L, which we refer to as DM25. This medium supports a stationary-phase density of about 5 x 107 cells per ml for the founding strain of E. coli B.
  • MG: Same basic composition as for DM liquid medium, except that we: add agar (as solidifying agent), increase the sugar concentration (so that colonies are robust), and sometimes use arabinose (instead of glucose). Glucose is used to examine the colonies on the standard minimal medium.
Protocol materials
ReagentMagnesium SulfateP212121
ReagentThiamine HClP212121
ReagentGlucoseP212121Catalog #Glucose
ReagentL-( )-ArabinoseMerck MilliporeSigma (Sigma-Aldrich)Catalog #A3256-500G
Reagent235-Triphenyltetrazolium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #T8877
ReagentPotassium phosphate (monobasic)P212121
ReagentTrisodium citrate dihydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S1804
ReagentAmmonium sulfateCatalog #97061-184
ReagentPotassium phosphate (dibasic)P212121
ReagentAgarMerck MilliporeSigma (Sigma-Aldrich)Catalog #A1296
ReagentAntifoam B EmulsionMerck MilliporeSigma (Sigma-Aldrich)Catalog #A5757-250ML
ReagentPotassium phosphate dibasic trihydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #P9666
ReagentBacto™ TryptoneThermo FisherCatalog #211705
ReagentBacto™ Yeast ExtractThermo FisherCatalog #212750
ReagentSodium ChlorideFisher ScientificCatalog # MK-7581-212
Troubleshooting
DM: Davis-Mingioli
To prepare Amount1 L of DM:

Weigh dry components:
a. Amount5.34 g of ReagentPotassium phosphate (dibasic)P212121 or Amount7 g of ReagentPotassium phosphate dibasic trihydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #P9666
b. Amount2 g of ReagentPotassium phosphate (monobasic)P212121
c. Amount1 g of ReagentAmmonium sulfateCatalog #97061-184
d. Amount0.5 g of ReagentTrisodium citrate dihydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S1804
Add distilled water to a final volume of Amount1 L
Autoclave using liquid program. Sterilization times are based on total volume:

Volume (ml)Time (min)
75-20020
200-50025
500-100030
1000-150035
1500-200040
>200060
Sterilization time

After autoclaving add the following stock solutions:
a. Amount1 mL of previously sterilizedReagentMagnesium SulfateP212121 at Concentration10 Mass / % volume s
b. Amount1 mL of filtered sterilized ReagentThiamine HClP212121 at Concentration0.2 Mass / % volume
If preparing DM-glucose, add this volume ofReagentGlucoseP212121Catalog #Glucose (separately autoclaved stock) at Concentration10 Mass / % volume , to get the final concentration desired:
per 1L of DMDMX[Glucose] (w/v)[Glucose] (mg/L)[Glucose] (M)
5 ml DM500 0.05% 500 mg/L 2.78 mM
250 µl DM25 0.0025% 25 mg/L 139 µM
20 ml DM2000 0.2% 2000 mg/L 11.1 mM
2.5 ml DM250 0.025% 250 mg/L 1.39 µM
10 ml DM1000 0.1% 1000 mg/L 5.55 mM
1 ml DM100 0.010% 100 mg/L 694 µM



Note
Remember: DMX = DM + X mg/L glucose. Glucose may no longer limit the final growth density above approximately DM1000. Remember: DMX = DM + X mg/L glucose.

Note
Final composition:
  • Sodium (Na+) = Concentration5.1 millimolar (mM)
  • Potassium (K+) = Concentration75.8 millimolar (mM)
  • Ammonium (NH4) = Concentration15.2 millimolar (mM)
  • Magnesium (Mg2+) = Concentration0.83 millimolar (mM)
  • Sulfate (SO42-) = Concentration8.41 millimolar (mM)
  • Phosphate (PO43-) = Concentration45.3 millimolar (mM)
  • Citrate = Concentration1.7 millimolar (mM)
  • (In DM25) Glucose = Concentration139 micromolar (µM)


TA: Tetrazolium Arabinose
To prepare Amount1.5 L of TA:

Prepare media base by combining in a 2L flask:
a. Amount15 g of ReagentBacto™ TryptoneThermo FisherCatalog #211705
b. Amount1.5 g of ReagentBacto™ Yeast ExtractThermo FisherCatalog #212750
c. Amount7.5 g of ReagentSodium ChlorideFisher ScientificCatalog # MK-7581-212
d. Amount24 g of ReagentAgarMerck MilliporeSigma (Sigma-Aldrich)Catalog #A1296
e. Amount1.5 mL of ReagentAntifoam B EmulsionMerck MilliporeSigma (Sigma-Aldrich)Catalog #A5757-250ML
Add distilled water to Amount1.3 L
Separately, prepare sugar solution by combining:
a. Amount15 g of ReagentL-( )-ArabinoseMerck MilliporeSigma (Sigma-Aldrich)Catalog #A3256-500G
b. Amount200 mL of distilled water

Note
Sugar could be substituted for any other sugar.

Autoclave both solutions, media base and sugar solution from Go to and Go to separatley and according to sterilization table in Go to .

Combine sterile solutions, media base and sugar solution for a total of Amount1.5 L
Add Amount1.5 mL of (filter sterilized and stored at Temperature4 °C )Reagent235-Triphenyltetrazolium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #T8877 at Concentration5 Mass / % volume

MG: Minimal glucose
To prepare Amount1 L of MG:

When making these plates it is necessary to prepare and autoclave the 3 main parts (salt solution, agar base, and sugar solution) separately. Compounds that inhibit growth are produced when agar and phosphate or phosphate and glucose are autoclaved together.
Critical

Prepare salt solution, combine:
a. Amount5.3 g of ReagentPotassium phosphate (dibasic)P212121
b. Amount2 g of ReagentPotassium phosphate (monobasic)P212121
c. Amount1 g of ReagentAmmonium sulfateCatalog #97061-184
d. Amount0.5 g of ReagentTrisodium citrate dihydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S1804
e. Amount400 mL of distilled water
Autoclave salt solution according to Go to
Prepare agar base by combining:
a. Amount16 g of ReagentAgarMerck MilliporeSigma (Sigma-Aldrich)Catalog #A1296
b. Amount1 mL of ReagentAntifoam B EmulsionMerck MilliporeSigma (Sigma-Aldrich)Catalog #A5757-250ML
c. Amount400 mL of distilled water
Autoclave agar base according to Go to
Prepare sugar solution by combining:
a. Amount4 g of ReagentGlucoseP212121Catalog #Glucose
b. Amount200 mL of distilled water
Note
Sugar could be substituted for any other sugar.

Autoclave sugar solution according to Go to
After the three parts have been autoclaved, combine the contents of the three flasks together while they are still warm add the following stock solutions:
a. Amount1 mL of ReagentMagnesium SulfateP212121 at Concentration10 Mass / % volume (separately autoclaved stock)
b. Amount1 mL of ReagentThiamine HClP212121 at Concentration0.2 Mass / % volume (filter sterilized stock)