Part:BBa_K1807001
Escherichia coli Exopolyphosphatase (PPX)
This part codes for the exopolyphosphatase (PPX) enzyme of Escherichia coli. PPX is able to release phosphate residues from the ends of a polyphosphate chain.
Usage and Biology
Exopolyphosphatase is an enzyme encoded by the PPX gene (Akiyama et al., 1993). Exopolyphosphatase cleaves phosphate residues from the termini of polyphosphate chains. Preferred substrate seems to be polyphosphate with approximately 500 residues long chain. ATP and short polyphosphate chains (around 15 residues) are either not a substrate at all or compete poorly with long polyphosphate.
Akiyama, M., E. Crooke, and A. Kornberg. "An exopolyphosphatase of Escherichia coli. The enzyme and its ppx gene in a polyphosphate operon." Journal of Biological Chemistry 268.1 (1993): 633-639.
iGEM York 2015 designed this protein generator part in order to test exopolyphosphatase's effect on phosphate uptake and storage of polyphosphate. This testing construct was based on the observations made in Keasling et al., 1998.
Keasling, J. D., Stephen J. Van Dien, and Jaya Pramanik. "Engineering polyphosphate metabolism in Escherichia coli: implications for bioremediation of inorganic contaminants." Biotechnology and bioengineering 58.2‐3 (1998): 231-239.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 872
Contributions
We have used albert stain to see if ppx has function.
Albert stain
Reagents:
Solution A:
Toluidine Blue 0.15g
Malachite Green 0.2g
Glacial acetic acid 1ml
95% ethanol 2ml
Distilled water 100ml
Solution B:
Iodine 2g
Potassium iodide 3g
Distilled water 300ml
Protocol:
1. Make a bacteria smear. Fire fixed.
2. Use solution A(80ml) dye it for 5 minutes
3. Wash under distilled water
4. Use solution B(80ml) dye it for 1 minute.
5. Wash under distilled water
6. After dry, use microscopy exam. The body of bacteria turn green and the metachromatic granules turn black blue.
The picture shows the WT stains darker than the ppx and ppn which means the ppn and ppx strain have lower phosphorus concentration. The photo is taken with same parameter at 1000×.Each group has 3 sample，they show the similar results with the picture here. The ppx and ppn strain are constructed on the pET28a(+) vector, also using IPTG to induce the expression. The result is acquired after 72 h induction.
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