Difference between revisions of "Part:BBa K2789021"

 
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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).
 
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).
 +
<br/>
 +
===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.
 +
<br/>
 +
 +
===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
 +
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 08:05, 17 October 2018


PPN(endopolyphosphotase)

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).

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.

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


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]