High-yield dual-buffered bacterial growth medium

Details High-yield dual-buffered bacterial growth medium High-yield dual-buffered bacterial growth medium

2001-04-04

2002-08-13

Lilling, Herbert J. (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S243000, C435S252800, C435S370000, C435S404000

Reexamination Certificate

active

06432691

ABSTRACT:

The present invention pertains to a high-yield, dual-buffered growth medium that supports the growth of bacterial cell cultures at higher densities than those achieved by conventional rich media.
The propagation of recombinant DNA in
E. coli
is a ubiquitous and necessary prerequisite for almost any endeavor in molecular biology, from cloning and sequencing of genes to ectopic expression of proteins. Many vectors have been used for this purpose, including plasmids, cosmids, and bacterial artificial chromosomes (BACs). Rich media such as Luria-Bertani (LB) broth, “Superbroth,” and “Terrific Broth” (TB) have been developed to enhance the normal growth of transformed bacterial cells.
See generally, F. M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, pp. 1.1.1 -1.1.3 (1997); J. Sambrook, E. F. Fritsch, and R. Maniatis, Molecular Cloning: A Laboratory Manual, 2
nd
ed., CSH Laboratory Press, pp. 1.21-1.23, A1-A3 (1989); K. D. Tartof and C. A. Hobbs, “Improved Media for Growing Plasmid and Cosmid Clones,”
Bethesda Res. Lab. Focus
vol. 9, p. 12 (1987). LB and its derivatives are the most commonly used, possibly because more complex recipes improve yield only slightly. In most cases, the moderate increase in yield does not justify the added time and effort to make the more complex media.
Cell density in liquid culture is limited by certain byproducts of metabolism. Growth of
E. coli
on excess glucose under aerobic conditions causes formation of acidic by-products, of which acetate is the most predominant. See H. W. Doelle et al., “Regulation of glucose metabolism in bacterial systems,”
Adv. Biochem. Eng.
vol. 23, pp.1-35 (1982); G. W. Luli and W. R. Strohl, “Comparison of growth, acetate production, and acetate inhibition of
Escherichia coli
strains in batch and fed-batch fermentations,”
Appl. Environ. Microbiol.
vol. 56, pp. 1004-1011 (1990)). High acetate concentrations can severely inhibit cell growth, genetic stability, and recombinant protein production. (G. W. Luli and W. R. Strohl,
Appl. Environ. Microbiol.
vol. 56, pp. 1004-1011 (1990)). While high cell density techniques have been developed for fed-batch fermentations (reviewed in G. L. Kleman and W. R. Strohl, “Developments in high cell density and high productivity microbial fermentation,”
Current Opinion in Biotechnology
vol. 5 pp. 180-186 (1994)), none have been developed for small-scale applications appropriate for the typical research laboratory.
U.S. Pat. Nos. 4,656,132 and 4,894,334 disclose a process for improving the yield of heterologous protein produced by recombinant bacteria by supplementing the nutrient medium with a water soluble alcohol and an amino acid mixture during the terminal phase of cultivation.
U.S. Pat. No. 5,223,418 discloses a method of improving the yield of heterologous proteins produced by recombinant
Streptomyces lividans
by using a complex nutrient medium supplemented with high concentrations of casamino acids (1-5%) and glucose (up to 3%).
U.S. Pat. No. 5,658,790 discloses a method to prepare a cell culture medium by using unit dose packaging of a dry concentrate of the medium. The formulations contain a single buffer, Tris, to maintain a neutral pH. Alternative embodiments contain low concentrations of glucose (0.4%) and casamino acids (0.6%).
In addition to external sources of carbon, nitrogen, and energy, bacteria can utilize an external source of ribonucleosides. Ribonucleotides in the growth medium can be hydrolyzed by periplasmic phosphatases, generating permeable ribonucleosides that can enter the cell and be used for RNA synthesis. (H. Zimmerman, “5′-Nucleotidase: Molecular Structure and Functional Aspects,”
Biochemical Journal
vol. 285, pp. 345-365 (1992)).
No known prior culture medium has a dual-buffer system or adds a source of free ribonucleotides.
There is a long felt need by those practicing recombinant DNA techniques for a high efficiency, inexpensive growth medium that can support high bacterial cell densities and high yields of plasmid DNA or of recombinant protein using equipment available in an ordinary research laboratory.
We have discovered a novel growth medium that supports much higher bacterial cell densities and, concomitantly, a much higher yield of plasmid than has been previously reported for small-scale applications. The high-yield growth medium contains two buffers with different pKa values and optionally includes an additional nucleotide source. On a unit volume basis,
E. coli
cultures grown in this medium have consistently produced 5-10 times, and sometimes up to 30 times, more recombinant plasmid than was produced in conventional rich media, paralleling the increase in cell density. This phenomenon is independent of
E. coli
host strain, DNA insert size, and plasmid copy number. The medium is economical, and high yields can be achieved using standard research laboratory equipment.


REFERENCES:
patent: 4071412 (1978-01-01), Eisenberg et al.
patent: 4656132 (1987-04-01), Ben-Bassat et al.
patent: 4894334 (1990-01-01), Ben-Bassat et al.
patent: 5223418 (1993-06-01), Arcuri et al.
patent: 5658790 (1997-08-01), Gautsch
patent: 6255099 (2001-07-01), Duttweiler et al.
Ausubel, F.M. et al., Current Protocols in Molecular Biology, John Wiley & Sons, vol. 1, pp. 1.1.1-1.1.3 (1995).
Doelle, H.W. et al., “Regulation of glucose metabolism in bacterial systems,”Adv. Biochem. Eng.vol. 23, pp. 1-35 (1982).
Duttweiler, H.M. et al., “Bacterial Growth Medium that Significantly Increases the Yield of Recombinant Plasmid,”BioTechniquesvol. 24, No. 3, pp. 438-444 (1998).
Kleman, G.L. and W. R. Strohl, “Developments in high cells density and high productivity microbial fermentation,”Current Opinion in Biotechnologyvol. 5, pp. 180-186 (1994).
Luli, G.W. and W. R. Strohl, “Comparison of Growth, Acetate Production; and Acetate Inhibition ofEscherichia coliStrains in Batch and Fed-Batch Fermentations,”Appl. Environ. Microbiol.vol. 56, No. 4, pp. 1004-1011 (1990).
Sambrook, J., E. F. Fritsch, and R. Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition, CSH Laboratory Press, pp. 1.21-1.23, A.1-A.3 (1989).
Tartof, K.D. and C. A. Hobbs, “Improved Media for Growing Plasmid and Cosmid Clones,”Bethesda Res. Lab. Focusvol. 9 No. 2, pp. 12 (1987).
Zimmerman, H., “5′-Nucleotidase: molecular structure and functional aspects,”Biochemical Journalvol. 285, pp. 345-365 (1992).

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