Vol. 3, No. 2 Products For Biomolecular NMR Download PDF version (1.49 MB)

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:

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.

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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.
 

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