Idea 6/11

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m (→Calcite/Calcium Phosphate Project (Idea 6/11))
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'''Aim'''
'''Aim'''
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To create a 3D structure in a defined area using bacteria which can precipitate out a calcium compound.
To create a 3D structure in a defined area using bacteria which can precipitate out a calcium compound.
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'''Possible Methods'''
'''Possible Methods'''
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If one colony lays down a substrate for a phosphatase enzyme, this could be the chemoattractant for a second colony to move in and start converting it into calcium phosphate.
 
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1. INPUT [UV] --> OUTPUT [glycerol phosphate]
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Initial colony makes phosphatase or urease in response to stimulus from UV, or absence of UV signal. The enzyme catalyses the conversion of calcium and phosphate/carbonate ions in the medium into calcium phosphate or calcium carbonate. An amplifier can be incorporated if the resulting amounts of 'hard' material need to be increased.
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2. INPUT [Glycerol phosphate and UV]--> OUTPUT [phosphatase]
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Input of glycerol phosphate and UV to colony two ensures phosphatase activity is only in the place where we want calcium phosphate to be deposited.
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OR, Simpler, initial colony makes phosphatase straight away. Could also use urease to make calcite precipitate.
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'''Problems'''
'''Problems'''
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How will the Calcium compound be restricted to only the areas activated? It could diffuse around the area and lose the original pattern.
How will the Calcium compound be restricted to only the areas activated? It could diffuse around the area and lose the original pattern.
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Can colony one be made to secrete a glycerol phosphate?
 
'''Parts Required'''
'''Parts Required'''
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'''Experiments Required'''
'''Experiments Required'''
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#Making sure E. coli can cause calcium compounds to precipitate. Urease hydrolyses urea to form ammonia and carbon dioxide.  
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#Finding out how long it takes.  
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Experiments for Idea 2.
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#Measuring the diffusion away from the defined area of the calcium precipitation.
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''Is calcium phosphate or calcite better at precipitating?''
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Urease vs. phosphatase.
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#Solutions of CaCl2 and either phosphate ion donor (eg/P2O5) or carbonate ion donor (sodium bicarbonate?). See what the quality of the product is and how long it takes to form.
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- Just mix together the two solutions. Can start at a low concentration of either substrate and work up to a higher one. Can do it both ways round to find a minimum concentration of both required to precipitate calcium compound.
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#Locate urease/phosphatase in E. coli that is suitable. (Alkaline phosphatase) Tests for conditions that will work. See how bacteria react with the ideal concentrations worked out in experiment one, (particularly for urease). CaCl2 concentrations, phosphate donor concentrations in medium. (Just test with genes coupled with constitutive promoters?) Can also see what happens when the medium, and if the medium becomes depleted of calcium or phosphate ions.
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End - Find a suitable ratio of calcium to phosphate ions in medium.
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#Urease increases pH, measure pH change caused by different densities of bacteria, find if the pH change produced by E. coli in a lawn is suitable for causing calcite to precipitate.  
[http://2006.igem.org/Ideas Ideas]
[http://2006.igem.org/Ideas Ideas]

Revision as of 14:21, 3 July 2006

Calcite/Calcium Phosphate Project (Idea 6/11)

Aim

To create a 3D structure in a defined area using bacteria which can precipitate out a calcium compound.

Background

Possible Methods

Initial colony makes phosphatase or urease in response to stimulus from UV, or absence of UV signal. The enzyme catalyses the conversion of calcium and phosphate/carbonate ions in the medium into calcium phosphate or calcium carbonate. An amplifier can be incorporated if the resulting amounts of 'hard' material need to be increased.

Problems If two colonies are used then colony two has to be able to get to colony one. Maybe colony one could be non-motile and colony two motile?

How will the Calcium compound be restricted to only the areas activated? It could diffuse around the area and lose the original pattern.

Parts Required

UV responsive sensor

glycerol-3-phosphate synthesis gene (periplasmic/outer membrane?)

glycerol-3-phosphatase/urease protein coding gene

Experiments Required

Experiments for Idea 2.

Is calcium phosphate or calcite better at precipitating?

Urease vs. phosphatase.

  1. Solutions of CaCl2 and either phosphate ion donor (eg/P2O5) or carbonate ion donor (sodium bicarbonate?). See what the quality of the product is and how long it takes to form.

- Just mix together the two solutions. Can start at a low concentration of either substrate and work up to a higher one. Can do it both ways round to find a minimum concentration of both required to precipitate calcium compound.

  1. Locate urease/phosphatase in E. coli that is suitable. (Alkaline phosphatase) Tests for conditions that will work. See how bacteria react with the ideal concentrations worked out in experiment one, (particularly for urease). CaCl2 concentrations, phosphate donor concentrations in medium. (Just test with genes coupled with constitutive promoters?) Can also see what happens when the medium, and if the medium becomes depleted of calcium or phosphate ions.

End - Find a suitable ratio of calcium to phosphate ions in medium.

  1. Urease increases pH, measure pH change caused by different densities of bacteria, find if the pH change produced by E. coli in a lawn is suitable for causing calcite to precipitate.


[http://2006.igem.org/Ideas Ideas]

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