Part:BBa_K2789021

PPN(endopolyphosphotase)

Description

This part can catalyze the degradation of PolyP(heterochromatic granules). It is different from PPX(exopolyphosphotase) that this protein can cut the PolyP from the internal site while PPX can only cut the Polyp from the ends of Poly P, this two protein together can dramatically release the phosphorus from the bacteria. This two proteins play the opposite effect from PPK(Polyphosphokinase).Since we chose the phosphorus as the recycling target this year. We cyclically express protein to absorb phosphorus in environment and release into our device.PPK works for accumulation and PPX worked for release. Since we wanted to collect phosphorus. The "release" step must be complete and thorough.PPN can help PPX to co-release phosphorus (improved about 10 times in speed). And we created PPN part this year and we used both staining method and quantitative measurement to verify that PPN can work!

Proof of the Expression

We have used pcr to confirm the fragment of the PPN and the PPX + RBS +PPN.

We also design a primer to fuse the PPN with the pET28a(+). By using the His-tag in frame with the vector, we purified the PPN protein and saw its expression in E.coli by SDS-PAGE.

1.Marker （the arrow indicates where the 55KDa protein is） 2.PPN with His-tag(1) 3.PPN with His-tag(2) 4.Control1 5.Control2

Albert stain

After that ,we use the Albert stain for metachromatic granules to see if the PPN works.
Reagent:
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.
Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 315
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1965
  
• 1000
  COMPATIBLE WITH RFC[1000]