Part:BBa_K2789032

PPX+RBS+PPN

This part can catalyze the degradation of PolyP(heterochromatic granules). It adds 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).

Fragment size

At first, we directly synthesize the ppx+rbs+ppn on the pET28a(+) vector and use the lac promoter on the vector. The plasmids extracted from the bacteria are used as templates to perform PCR and the results show the fragment is right about 4000bps.

Function proof

We add 100μL WT and the transformed BL21 separately into 100ml LB culture and incubate the bacteria at 37°C with 200rpm in about 8 hours. At the same time，we arbitrarily extract the culture to measure the OD600 and no distinct difference occurs(OD600WT:0.8 OD600test group:0.9). The IPTG is added after the sampling to 0.5mM and incubation continues.
We use the kit manufactured by HANGZHOU LOHAND BIOLOGICAL Co. Ltd. to measure the phosphorus concentration in the media after 16h. Before that, the culture is measured at 600nm to test the concentration of bacteria and centrifuged at 10,000×g for 3 minutes to clear the most bacteria. The supernatant is diluted with 49times water to make the phosphorus suitable to measure. The reaction result shows below:

From the picture 6, we can see obvious differences between the test group and WT. The more phosphorus in the sample, the bluer it is. Thus, the concentration P in the test group is higher than the control. However, as we measured the OD600 in the media, the result is largely due to the concentration of the bacteria instead of the ppx with ppn. The graph shows here is calculated by (C_1-C_2)/OD .C1 is the phosphorus concentration right before the induction .C2 is the phosphorus concentration right after the 16h induction. OD is the OD600 value which indicates the concentration of the bacteria. From the result, we think that the induction may be so strong that the protein produce is at a very high level which is not good for the bacteria growth. But if we compare the induced BL21 and the not induced group，we can still see a difference.

Conclusion

A 9% decrease compared with not-induced group and 2% decrease compared with the WT is seen by the figure below.

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 2094
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 3744
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 872