Difference between revisions of "Part:BBa K1807009"
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We took ppk1 in Citrobacter freundii ATCC 8090 as a comparison and validated its activity through the experiment. We found that like in  Citrobacter freundii ATCC 8090, ppk1 can also produce polyP in the E.coli, which proved our hypothesis.After incubation in the polyphosphorous culture medium for 20 min, we found that polyP was produced by PPK1, which was proved by ashing and spectrum.
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Polyphosphate kinase(PPK) polymerizes the terminal phosphate of ATP to a long chain of polyphosphate(polyP) in a freely reversible reaction. Here polyphosphate kinase was purified and characterized by SDS-PAGE. Bands of 69kDa showed the existence of PPK in the Escherichia coli.
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<p>We also measured the curve of OD value CPVM in PA medium. Here is our result:
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[[File:T--Nanjing-China--ppk1.png|800px|thumb|center|Figure1 SDS-PAGE analysis of PPK]]
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[[File:T--Nanjing-China--CPVM1.png|800px|thumb|center|Figure 1）OD measurement of CPVM]]  
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We compared results of CPVM with results of EPVM, and proved the activity of ppk1 in Citrobacter freundii ATCC 8090.
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Additionally, efficiency of polyP synthesis by PPK was measured at 1mM ATP. As shown in the diagram, polyP synthesis was linear with time for about 40 min. However, if the concentration of ATP is lower than 5 μM, there is virtually no synthesis of polyP for 20 min. 
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[[File:T--Nanjing-China--ppk11.png|800px|thumb|center|Figure2 Time course of polyP synthesis at 1mM ATP]]  
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<h2>iGEM2019_Nanjing China Experiment</h2>
 
<h2>iGEM2019_Nanjing China Experiment</h2>

Revision as of 07:56, 10 October 2021


Polyphosphate kinase enzyme from Escherichia coli

Coding sequence for the Polyphosphate enzyme (PPK) from Escherichia coli.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 317
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1671
  • 1000
    COMPATIBLE WITH RFC[1000]


iGEM2021_Nanjing-China Experiment

Group: Nanjing-China 2021
Author: Hao Yin

Polyphosphate kinase(PPK) polymerizes the terminal phosphate of ATP to a long chain of polyphosphate(polyP) in a freely reversible reaction. Here polyphosphate kinase was purified and characterized by SDS-PAGE. Bands of 69kDa showed the existence of PPK in the Escherichia coli.

Figure1 SDS-PAGE analysis of PPK

Additionally, efficiency of polyP synthesis by PPK was measured at 1mM ATP. As shown in the diagram, polyP synthesis was linear with time for about 40 min. However, if the concentration of ATP is lower than 5 μM, there is virtually no synthesis of polyP for 20 min.

Figure2 Time course of polyP synthesis at 1mM ATP

iGEM2019_Nanjing China Experiment

This year our team develops a simple solo medium-copy plasmid-based polyphosphate kinase (PPK1) overexpression strategy for achieving maximum intracellular polyphosphate accumulation, so the data can provide some reference to this part.

We test supernatant Pi concentration and optical density of Ecoil(DH5a) and DH5a-MDPP in Synthetic municipal wastewater(SMW).

Ps: SMW means Synthetic municipal wastewater

DH5a-MDPP means solo medium-copy DH5a ppk in DH5a

Figure 1）supernatant Pi concentration in SMW

Figure 2）optical density of Ecoil in SMW