Media comprising a glutamine biosensor and methods of use thereof

• US 9,279,139 B2
• Filed: 06/20/2012
• Issued: 03/08/2016
• Est. Priority Date: 06/21/2011
• Status: Active Grant

• Current Assignee: University of Guelph
• Original Assignee: University of Guelph
• Inventors: Raizada, Manish, Tessaro, Michael, Shehata, Hanan Reda Hassan Elsayed
• Primary Examiner(s): GITOMER, RALPH J
• Application Number:
  US14/127,863
• Publication Number:
  US 20140329271A1
• Time in Patent Office: 1,357 Days
• Field of Search: 435/34, 435/29, 435/4, 435/419
• US Class Current: 1/1
• CPC Class Codes: C12N 9/0069 acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
• CPC Class Codes: C12N 9/93 Ligases (6)
• CPC Class Codes: C12Q 1/02 involving viable microorganisms
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: C12Q 1/045 Culture media therefor
• CPC Class Codes: C12Q 1/66 involving luciferase
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters
• CPC Class Codes: C12Y 113/12007 Photinus-luciferin 4-monooxygenase (ATP-hydrolysing) (1.13.12.7), i.e. firefly-luciferase
• CPC Class Codes: C12Y 603/01002 Glutamate-ammonia ligase (6.3.1.2)
• CPC Class Codes: G01N 33/6812 Assays for specific amino acids

Abstract

An essentially glutamine-free medium comprising a whole-cell glutamine biosensor, comprising a glutamine auxotrophic E. coli and a Lux reporter gene, for detecting glutamine in an analyte. The medium is useful in methods to detecting, screening, identifying and selecting nitrogen-fixing microbes and in methods of determining the nitrogen state of plants, plant products and soil.

Claims

1. An essentially glutamine-free medium comprising a glutamine biosensor for detecting glutamine in an analyte, wherein the glutamine biosensor is a whole cell E. coli GlnLux glutamine biosensor.

2. The medium of claim 1, wherein the glutamine biosensor comprises a bacterial cell that is a glutamine auxotroph.

3. The medium of claim 2, wherein the bacterial cell is E. coli.

4. The medium of claim 2, wherein glutamine synthetase is down-regulated in the bacterial cell as compared to a wild-type bacterial cell.

5. The medium of claim 2, wherein the bacterial cell comprises a reporter gene that is expressed in the presence of glutamine.

6. The medium of claim 5, wherein the reporter gene is lux.

7. The medium of claim 1, wherein the growth medium is Lysogeny Broth (LB), M9 medium, agar, or combinations thereof, and optionally comprises casamino acids and/or one or more protease inhibitors.

8. The medium of claim 7, wherein the medium comprises casamino acids in an amount of about 0.5% v/v, and protease inhibitors in an amount of about 1% v/v.

9. The medium of 1, wherein the medium is liquid, solid, or semi-solid, and optionally wherein the glutamine biosensor is diluted up to about 1000-fold.