Idea 6/11
From 2006.igem.org
Contents |
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.
- 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.
- 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.
- 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.
