Team:Newcastle/Urease

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==Biochemical network==
==Biochemical network==
[[Image:Newcastle_Arginine_and_Ornithine_Degradation.png|600px]]
[[Image:Newcastle_Arginine_and_Ornithine_Degradation.png|600px]]
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Taken from [http://seed-viewer.theseed.org/seedviewer.cgi?page=Subsystems&subsystem=Arginine_and_Ornithine_Degradation&organism=224308.1 SEED]
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[[Image: rocFalan.jpeg|600px]]
[[Image: rocFalan.jpeg|600px]]

Revision as of 13:01, 5 September 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 are looking at another strategy, increasing 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. We believe that in turn the urea itself will increase urease production. By targeting this pathway we will hopefully be able to activate additional unknown pathways and enzymes that are involved in urease production.

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 Urease BioBrick.

SR1 BioBrick

SR1 is a small untranslated regulatory RNA from the Bacillus subtilis genome. It acts as an antisense RNA, base pairing with ahrC mRNA and inhibiting its translation. ahrC mRNA encodes AhrC, 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.

..diagram..

rocF BioBrick

..diagram..

Composite urease BioBrick

..diagram..

Biochemical network

Newcastle Arginine and Ornithine Degradation.png

Taken from SEED

RocFalan.jpeg

Computational model

...

ModelrocFsr1.png

Cloning strategy

Characterisation

See [1] [2] [3]

Characterisation of rocF The aim of this experiment is to determine whether the rocF BioBrick increases arginase production.

  • Materials Required
  • Plate consisting of Bacillus subtilis 168 colonies.
  • Flame (streaking) Loop
  • LB media consisting arginine and ampicillin
  • Auto pipette
  • Bursen Burner
  • Universal Tube
  • Procedure

Perform the experiment using aseptic technique. Transfer B. subtilis 168 colonies into universal tubes containing 5 ml of LB media and allowed to grow overnight at 37° C. Transfer 1 ml of the overnight culture to another universal tube containing 4 ml of the following media:

  1. Control (1) - LB media
  2. Control (2) - LB media with 10 mM of arginine
  3. Control (3) - LB media plus B. subtilis 168
  4. Test (1) - LB media with 10 mM of arginine plus B. subtilis 168
  5. Test (2) - LB media with 10 mM of arginine plus B. subtilis 168
  • Incubate the culture at 37° C with shaking.
  • Record the pH at every 30 min interval. Use 20 ul of the culture and measure the pH.

Expected results Arginase breaks down arginine to orthinine. Increasing production of arginase in the cells will cause a faster breakdown of arginine, thus faster changes in pH level.

  1. Control (1) - No change in pH
  2. Control (2) - No change in pH
  3. Control (3) - Increase in pH, however will be lower than test 1 and test 2.
  1. Test (1) - Increase in pH
  2. Test (2) - Increase in pH




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 Cement and Concrete Research 40 (2010) 157–166

Use of bacteria to repair cracks in concrete Kim Van Tittelboom a, Nele De Belie a,⁎, Willem De Muynck a,b, Willy Verstraete b


Expression of the rocDEF Operon Involved in Arginine Catabolism in Bacillus subtilis Rozenn Gardan, Georges Rapoport and Michel Debarbouille J. Mol. Biol. (1995) 249, 843–856


Expression of the Bacillus subtilis ureABC Operon Is Controlled by Multiple Regulatory Factors Including CodY,GlnR, TnrA, and Spo0H


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