Jul 13, 2023

LTEE Media Recipes V.1

DOI
https://dx.doi.org/10.17504/protocols.io.n92ldpy5ol5b/v1
  • 1University of Texas at Austin;
  • 2The University of Texas at Austin
  • the-ltee
Jeffrey E Barrick
Icon indicating open access to content
QR code linking to this content
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 26, 2022
Last Modified: July 13, 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
Magnesium SulfateP212121
Thiamine HClP212121
GlucoseP212121Catalog #Glucose
L-( )-ArabinoseMerck MilliporeSigma (Sigma-Aldrich)Catalog #A3256-500G
235-Triphenyltetrazolium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #T8877
Potassium phosphate (monobasic)P212121
Trisodium citrate dihydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S1804
Ammonium sulfateCatalog #97061-184
Potassium phosphate (dibasic)P212121
AgarMerck MilliporeSigma (Sigma-Aldrich)Catalog #A1296
Antifoam B EmulsionMerck MilliporeSigma (Sigma-Aldrich)Catalog #A5757-250ML
Potassium phosphate dibasic trihydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #P9666
Bacto™ TryptoneThermo FisherCatalog #211705
Bacto™ Yeast ExtractThermo FisherCatalog #212750
Sodium ChlorideFisher ScientificCatalog # MK-7581-212
DM: Davis-Mingioli
To prepare 1 L of DM:

Weigh dry components:
a. 5.34 g of Potassium phosphate (dibasic)P212121 or 7 g of Potassium phosphate dibasic trihydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #P9666
b. 2 g of Potassium phosphate (monobasic)P212121
c. 1 g of Ammonium sulfateCatalog #97061-184
d. 0.5 g of Trisodium citrate dihydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S1804
Add distilled water to a final volume of 1 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. 1 mL of previously sterilizedMagnesium SulfateP212121 at 10 Mass / % volume s
b. 1 mL of filtered sterilized Thiamine HClP212121 at 0.2 Mass / % volume
If preparing DM-glucose, add this volume ofGlucoseP212121Catalog #Glucose (separately autoclaved stock) at 10 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+) = 5.1 millimolar (mM)
  • Potassium (K+) = 75.8 millimolar (mM)
  • Ammonium (NH4) = 15.2 millimolar (mM)
  • Magnesium (Mg2+) = 0.83 millimolar (mM)
  • Sulfate (SO42-) = 8.41 millimolar (mM)
  • Phosphate (PO43-) = 45.3 millimolar (mM)
  • Citrate = 1.7 millimolar (mM)
  • (In DM25) Glucose = 139 micromolar (µM)


TA: Tetrazolium Arabinose
To prepare 1.5 L of TA:

Prepare media base by combining in a 2L flask:
a. 15 g of Bacto™ TryptoneThermo FisherCatalog #211705
b. 1.5 g of Bacto™ Yeast ExtractThermo FisherCatalog #212750
c. 7.5 g of Sodium ChlorideFisher ScientificCatalog # MK-7581-212
d. 24 g of AgarMerck MilliporeSigma (Sigma-Aldrich)Catalog #A1296
e. 1.5 mL of Antifoam B EmulsionMerck MilliporeSigma (Sigma-Aldrich)Catalog #A5757-250ML
Add distilled water to 1.3 L
Separately, prepare sugar solution by combining:
a. 15 g of L-( )-ArabinoseMerck MilliporeSigma (Sigma-Aldrich)Catalog #A3256-500G
b. 200 mL of distilled water

Note
Sugar could be substituted for any other sugar.

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

Combine sterile solutions, media base and sugar solution for a total of 1.5 L
Add 1.5 mL of (filter sterilized and stored at 4 °C )235-Triphenyltetrazolium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #T8877 at 5 Mass / % volume

MG: Minimal glucose
To prepare 1 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.

Prepare salt solution, combine:
a. 5.3 g of Potassium phosphate (dibasic)P212121
b. 2 g of Potassium phosphate (monobasic)P212121
c. 1 g of Ammonium sulfateCatalog #97061-184
d. 0.5 g of Trisodium citrate dihydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S1804
e. 400 mL of distilled water
Autoclave salt solution according to
Prepare agar base by combining:
a. 16 g of AgarMerck MilliporeSigma (Sigma-Aldrich)Catalog #A1296
b. 1 mL of Antifoam B EmulsionMerck MilliporeSigma (Sigma-Aldrich)Catalog #A5757-250ML
c. 400 mL of distilled water
Autoclave agar base according to
Prepare sugar solution by combining:
a. 4 g of GlucoseP212121Catalog #Glucose
b. 200 mL of distilled water
Note
Sugar could be substituted for any other sugar.

Autoclave sugar solution according 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. 1 mL of Magnesium SulfateP212121 at 10 Mass / % volume (separately autoclaved stock)
b. 1 mL of Thiamine HClP212121 at 0.2 Mass / % volume (filter sterilized stock)