Vol. 3, No. 2
Products For
Biomolecular NMR

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Page 1: Intro / Labeled Products for Cell Free Synthesis / Products for Peptide Synthesis / Products for Minimal Media
Page 2: Labeled Complex Growth Media / Buffers and Reagents / a-Keto Acids / Labeled Nucleotides

Labeled Complex Growth Media

Fermentation with ISOGRO™-13C Powder Supplementation

Andrew Clark University of Alabama Huntsville

E. coli (Rosetta™(DE3)pLysS, Novagen®) was grown in a fermentor (BioFlo 3000®, New Brunswick) with a 2-liter vessel containing minimal media with ISOGRO™-13C as a supplement in a quantity which is 5 % of its recommended usage. The composition of the minimal media, as adapted from Molecular Cloning (Maniatis et al.), is as follows: Na2HPO4 (12.8 g/L), KH2PO4 (3 g/L), NaCl (0.5 g/L), MgSO4 (0.001 M), CaCl2 (5E-5 M), glucose (2 g/L), NH4Cl (1 g/L), and ampicillin (60 mg/ml).

First of all, a 200-ml minimal media culture was grown in a shaker flask with and without 0.1 g of ISOGRO™-13C (5 % of its recommended usage) to assess its effectiveness as a supplement in the shaker flask before implementing it into fermentation. The culture with the ISOGRO™ absent grew to a maximal optical density at 600 nm (OD) of 1.4, whereas the culture with the ISOGRO™ present grew to a maximal OD of 2.5.

With this information in hand, I set out to assess the effectiveness of using ISOGRO™ as a supplement in the fermentor. The recombinant protein I am working with is encoded by the pET-3b vector (Novagen®) which contains the T7 promoter, and so its expression is induced by the addition of IPTG (Sigma # I 6758). It had already been previously established that there was a direct correlation between the maximal OD reached in the fermentor and the quantity of protein rendered upon preparation of the cells and purification.

It had also been established that the maximal OD could be increased by adding the glucose gradually instead of all at the beginning. This led me to speculate as to whether the maximal OD could be increased by adding the ISOGRO™ gradually. The results of all of my fermentation runs involving ISOGRO™ and one control experiment are presented in the following table:

OD Experiment
2.4 5 ml of 20 % glucose (m/V) added at beginning and 15 ml of 20 % glucose added gradually in 5-ml increments. ISOGRO™ was absent (control).
3.2 5 ml of 20 % glucose and 0.9 g ISOGRO™-13C added at beginning and 15 ml of 20 % glucose added gradually in 5-ml increments.
2.8 5 ml of 20 % glucose and 12.5 ml of ISOGRO™-13C (1 g/50 ml) added at beginning and 15 ml of 20 % glucose and 37.5 ml of ISOGRO™-13C added gradually in 5- and 12.5-ml increments, respectively.
3.0 5 ml of 20 % glucose and 50 ml of ISOGRO™-13C (1 g/50 ml) added at beginning and 15 ml of 20 % glucose added gradually in 5-ml increments.


It appears that whereas the OD increased when the glucose was added gradually, the OD actually increased when the ISOGRO™ was added all at once at the beginning. There was a significant increase in the maximal OD reached due to the addition of the ISOGRO™ as a supplement instead of as the sole source of nutrients. This optimization of high-density E. coli fermentation will definitely lead to an increase in the yield of protein from the cultures grown and improve our laboratory's capability to produce large quantities of isotope labeled proteins for Biomolecular NMR studies.

 

 


Typical Procedure for Growing E. coli Using ISOGRO™ Powder

To prepare 100mL ISOGRO™ medium:

  1. Dissolve 1.0g of ISOGRO™ powder in about 90mL of Millipored® water.
  2. Make stock solutions of the following salts and use the quantities indicated in the medium preparation:
    Salt Conc. of Stock Soln. Qty./100mL medium
    K2HPO 100g/L 1.8mL
    KH2PO4 50g/L 2.8mL
    MgSO4 50g/L 2.0mL
    CaCl2H2O 37g/L 30µL

  3. Adjust pH to 7.0 with NaOH and bring solution up to 100mL with Millipored® water.
  4. Pass the solution through a 0.22mM filter and transfer the filtrate to an autoclaved shaker flask (for example: 50mL medium in a 500mL flask).
  5. The culture is inoculated with a loop of E. coli which has been maintained on a nutrient agar slant.
  6. Shake the culture flask in a 37°C water bath.
  7. The absorbance of the culture is measured at 600nM with a 1:3 dilution into water.

Note: Researcher's specific expression applications vary, so our preparation should serve as a guideline.

 

Cat. No. Product Name Atom %
60,686-3 ISOGRO™-13C Powder - Growth Medium 99
61,672-9 ISOGRO™-D Powder - Growth Medium 97-99
60,687-1 ISOGRO™-15N Powder - Growth Medium 98
60,683-9 ISOGRO™-13C,15N Powder - Growth Medium 99 13C; 98 15N
60,830-0 ISOGRO™-15N,D Powder - Growth Medium 98 15N; 97-99 D
60,829-7 ISOGRO™-13C,15N,D Powder - Growth Medium 99 13C; 98 15N; 97-99 D

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Buffers and Reagents
Cat. No. Product Name Atom %
15,178-5 Acetic Acid-d4 99.5
17,657-5 Ammonium-d4 Bromide 98
17,567-6 Ammonium-d4 Chloride 98
17,670-2 Ammonium-d4 Deuteroxide (25% in D2O) 99
48,835-6 Butanedioic Acid-d6 98
48,553-5 DL-1,4-Dithiothreitol-d10 98
48,561-6 Dodecylphosphorylcholine-d38 98
48,937-9 Ethylenediaminetetraacetic-d12 Acid 98
42,622-9 Formic Acid-d2 (95% in D2O) 98
17,583-8 Glycine-d5 98
36,602-1 Imidazole-d4 98
61,522-6 2-Mercaptoethanol-d6 96
37,384-2 Sodium Formate-d 99
45,185-1 Sodium Lauryl-d25 Sulfate 98
44,910-5 TRIS-d11 (crystalline) [Tris(hydroxymethyl)aminomethane] 99
48,624-8 TRIS-d11 (~1M solution in D2O) 99
44,749-8 Glycerol-d8 98
45,452-4 Glycerol-1,1,2,3,3-d5 98
48,947-6 Glycerol-13C3 99
27,717-7 Methyl-13C Alcohol (< 5% 18O) (Methanol) 99
28,201-4 Sodium Acetate-13C2 99
17,607-9 Sodium Acetate-d3 99
29,911-1 Sodium Acetate-13C2, d3 99 13C; 99 D

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a-Keto Acids

Research has shown that isotopically labeled ketobutyric and pyruvic acids can be used as constituents in modified E. coli growth media to produce bio-synthesized proteins with unique Cd2H and CDH2 groups. These selectively labeled methyl groups greatly reduce overlap in INEPT Biomolecular NMR, a desirable property for spectral interpretation and protein structure elucidation. ISOTEC™ offers the following isotopically labeled ketobutyric and pyruvic acids. If you require a related compound not listed below, please contact us for a custom quote.

References: R. Ishima, J.M. Louis, D. Torchia. Journal of Biomolecular NMR 21:167-171, 2001 N.K. Goto, K.H. Gardner, G.A. Mueller, R.C. Willis, L.E. Kay. Journal of Biomolecular NMR 13:369-374, 1999.
 

Cat. No. Product Name Atom %
57,134-2 2-Ketobutyric-4-13C Acid, Sodium Salt•xH2O (97% CP) 99
58,927-6 2-Ketobutyric-4-13C,3,3-d2 Acid, Sodium Salt•H2O (98% CP) 99 13C; 98 D
60,754-1 2-Ketobutyric Acid-13C4,3,3-d2, Sodium Salt•xH2O (98% CP) 99 13C; 97-98 D
60,753-3 2-Ketobutyric-4-13C,3,3,4,4,4-d5 Acid, Sodium Salt•xH2O
(98% CP)
99 13C; 98 Cd2;
50-70 Cd3
57,133-4 2-Keto-3-methyl-13C-butyric-4-13C Acid, Sodium Salt 99
58,906-3 2-Keto-3-methyl-13C-butyric-4-13C,3-d1 Acid, Sodium Salt 99 13C; 98 D
58,490-3 2-Keto-3-methyl-d3-butyric-4-13C Acid, Sodium Salt 99 13C; 98 D
59,641-8 2-Keto-3-methyl-d3-butyric Acid-1,2,3,4-13C4, Sodium Salt 99 13C; 98 D
60,756-8 2-Keto-3-methylbutyric Acid-13C5, 3-d1, Sodium Salt 99 13C; 98 D
49,073-3 Sodium Pyruvate-3-13C 99
60,848-3 Sodium Pyruvate-3-13C-3,3,3-d3 99 13C; 50-60 D

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Labeled Nucleotides
13C,15N-Ribonucleotides (minimum 90% chemical purity), supplied as sodium salts
Cat. No. Product Name Atom %
60,835-1 Adenosine-13C10,15N5 5'-triphosphate 99 13C; 98 15N
60,837-8 Cytidine-13C9,15N3 5'-triphosphate 99 13C; 98 15N
60,838-6 Guanosine-13C10,15N5 5'-triphosphate 99 13C; 98 15N
60,839-4 Uridine-13C9,15N2 5'-triphosphate 99 13C; 98 15N
13C,15N- Deoxyribonucleotides, supplied as sodium salts
Cat. No. Product Name Atom %
60,840-8 2'-Deoxyadenosine-13C10,15N5 5'-triphosphate 99 13C; 98 15N
60,841-6 2'-Deoxycytidine-13C9,15N3 5'-triphosphate 99 13C; 98 15N
60,842-4 2'-Deoxyguanosine-13C10,15N5 5'-triphosphate 99 13C; 98 15N
60,843-2 2'-Deoxythymidine-13C10,15N2 5'-triphosphate 99 13C; 98 15N