Team:Newcastle/Urease

From 2010.igem.org

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#Too see charcaterization of ''rocF'', please refer to the cloning strategy of ''rocF''.
#Too see charcaterization of ''rocF'', please refer to the cloning strategy of ''rocF''.
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==References==
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Biosynthesis of Active Bacillus subtilis Urease in the Absence of Known Urease Accessory Proteins JOURNAL OF BACTERIOLOGY, Oct. 2005, p. 7150–7154 Jong Kyong Kim, Scott B. Mulrooney, and Robert P. Hausinger
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#Kim JK, Mulrooney SB, and Hausinger RP. 2005. "''Biosynthesis of Active Bacillus subtilis Urease in the Absence of Known Urease Accessory Proteins''". Journal of Bacteriology.p.7150–7154.
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Cement and Concrete Research 40 (2010) 157–166
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Use of bacteria to repair cracks in concrete Kim Van Tittelboom a, Nele De Belie a,⁎, Willem De Muynck a,b, Willy Verstraete b
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#Tittelboom KV, Belie ND, Muynck WD, Verstraete W. 2010. "''Use of bacteria to repair cracks in concrete''". Cement and Concrete Research. 40. p.157–166.
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Expression of the rocDEF Operon Involved in Arginine Catabolism in Bacillus subtilis
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#Gardan R, Rapoport G and Debarbouille M. 1995. "''Expression of the rocDEF Operon Involved in Arginine Catabolism in Bacillus subtilis''". Journal of Molecular Biology. 249, p.843–856.
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Rozenn Gardan, Georges Rapoport and Michel Debarbouille J. Mol. Biol. (1995) 249, 843–856
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Expression of the Bacillus subtilis ureABC Operon Is Controlled
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by Multiple Regulatory Factors Including CodY,GlnR, TnrA, and Spo0H
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Revision as of 12:50, 9 October 2010

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Contents

Calcium carbonate precipitation via urease expression

Bacillus subtilis produce urease, which catalyses the hydrolysis of urea into ammonium and carbonate (CO32-). Since the cell walls of the bacteria are negatively charged, they draw cations from the environment, including Ca2+, to deposit on their cell surface. The Ca2+ ions subsequently react with the CO32- ions, leading to the precipitation of CaCO3 at the cell surface.

In order for B. subtilis to fill up cracks in concrete, enhanced production of calcium carbonate must be achieved: we need to up-regulate urease production.

Previous experiments involving up-regulating ureA, ureB and ureC in B. subtilis have not lead to an increase in urease production. This could be due to yet unidentified genes that are involved in the process. Therefore, we looked for another strategy.

Flux balance analysis

In order to identify pathways which indirectly lead to urea hydrolysis we performed flux balance analysis using the COBRA Matlab Toolbox and a model of the core B. subtilis 168 metabolic network.

What flux balance is....

By ..setting objective to maximise urease activity.. we were able to identify the arginine biosynthesis and catabolism pathways as a potential targets.

........Results, Matlab file........

By increasing arginine and arginase production we can increase urea hydrolysis indirectly. Arginase breaks down arginine to urea and ornithine, leading to an increase of urea inside the cell. We believe that in turn the urea itself will increase urease production.

BioBricks

We plan to produce two BioBricks, SR1, which will enhance arginine production, and rocF, which will enhance arginase production. These will be combined into a composite urea/urease BioBrick.

Arginine BioBrick

Newcastle IPTG-inducible L-arginine.png

SR1 is a small untranslated regulatory mRNA from the Bacillus subtilis genome. It acts as an antisense RNA to ahrC mRNA thereby inhibiting its translation. ahrC mRNA encodes AhrC protein, which represses arginine biosynthesis and positively regulates arginine catabolism.

Transcription of SR1 results in an increase in arginine biosynthesis and a decrease in arginine catabolism thus overall arginine level increases within the cell.

This is part BBa_K302013 on the parts registry.

Arginase BioBrick

IPTG-inducible arginase.png

The rocF gene codes for the enzyme arginase, which breaks arginine into ornithine and urea. This is part BBa_K302014 on the parts registry.

Composite urea/urease BioBrick

IPTG-inducible urea urease.png

Part BBa_K302015 on the parts registry combines the above two BioBricks. The part increases urea hydrolysis indirectly, by increasing arginine and arginase production. Arginase breaks down arginine to urea and ornithine, leading to an increase of urea inside the cell. In turn the urea itself leads to urease production. Urease breaks urea into ammonia and carbonate ions and the carbonate ions are then transported to the extracellular face of the cell membrane.

Biochemical network

Newcastle Arginine and Ornithine Degradation.png

Taken from SEED

Alan showing urease pathway

Computational model

...

ModelrocFsr1.png

Cloning strategy

Characterisation

See [1] [2] [3]

  1. Too see charcaterization of SR1, please refer to the cloning strategy of SR1.
  2. Too see charcaterization of rocF, please refer to the cloning strategy of rocF.

References

  1. Kim JK, Mulrooney SB, and Hausinger RP. 2005. "Biosynthesis of Active Bacillus subtilis Urease in the Absence of Known Urease Accessory Proteins". Journal of Bacteriology.p.7150–7154.
  1. Tittelboom KV, Belie ND, Muynck WD, Verstraete W. 2010. "Use of bacteria to repair cracks in concrete". Cement and Concrete Research. 40. p.157–166.


  1. Gardan R, Rapoport G and Debarbouille M. 1995. "Expression of the rocDEF Operon Involved in Arginine Catabolism in Bacillus subtilis". Journal of Molecular Biology. 249, p.843–856.
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