Difference between revisions of "Part:BBa K1807009"
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===Experimental characterization added by CPU-Nanjing 2022 TEAM===
 
===Experimental characterization added by CPU-Nanjing 2022 TEAM===
 
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Team Cpu-Nanjing has added new experimental characterization on this existing Part.
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Team CPU-Nanjing has added new experimental characterization on this existing Part.
  
 
Polyphosphate kinase (PPK) catalyzes the synthesis of polyphosphate (polyP). PolyP is a polyanionic biopolymer and generally stored in cells in the form of polyP granules. Theoretically, the presence of intracellular polyP granules can be observed with light microscopy after the cells had been stained with the cationic dye, such as toluidine blue. To test this, we overexpressed this Part using plasmid pBBR1MCS2 in E. coli K12 and examined polyP granules in toluidine blue stained cells.
 
Polyphosphate kinase (PPK) catalyzes the synthesis of polyphosphate (polyP). PolyP is a polyanionic biopolymer and generally stored in cells in the form of polyP granules. Theoretically, the presence of intracellular polyP granules can be observed with light microscopy after the cells had been stained with the cationic dye, such as toluidine blue. To test this, we overexpressed this Part using plasmid pBBR1MCS2 in E. coli K12 and examined polyP granules in toluidine blue stained cells.
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After staining, polyP can be easily visualized as blue-purple granules.
 
After staining, polyP can be easily visualized as blue-purple granules.
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 +
===Improvement made by CPU-Nanjing 2022 TEAM===
 +
 +
CPU-Nanjing has improved the existing part by site-directed mutagenesis of two amino acids and developed a highly active polyphosphate kinase. Number of the improved part is BBa_K4257000.
  
 
==iGEM2021_Nanjing-China Experiment==
 
==iGEM2021_Nanjing-China Experiment==

Revision as of 11:38, 27 September 2022


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]


Experimental characterization added by CPU-Nanjing 2022 TEAM

Team CPU-Nanjing has added new experimental characterization on this existing Part.

Polyphosphate kinase (PPK) catalyzes the synthesis of polyphosphate (polyP). PolyP is a polyanionic biopolymer and generally stored in cells in the form of polyP granules. Theoretically, the presence of intracellular polyP granules can be observed with light microscopy after the cells had been stained with the cationic dye, such as toluidine blue. To test this, we overexpressed this Part using plasmid pBBR1MCS2 in E. coli K12 and examined polyP granules in toluidine blue stained cells.

CPU-Nanjing-Parts-PPK-1.png
Figure 1. Microscopic observation of polyP granules in E. coli K12/PPK. Intracellular polyP granules appear blue-purple. Scale bar, 2 μm.

After staining, polyP can be easily visualized as blue-purple granules.

Improvement made by CPU-Nanjing 2022 TEAM

CPU-Nanjing has improved the existing part by site-directed mutagenesis of two amino acids and developed a highly active polyphosphate kinase. Number of the improved part is BBa_K4257000.

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

Reference: Kyunghye Ahn and Arthur Kornberg. Polyphosphate Kinase from Escherichia coli[J]. The Journal of Biological Chemistry, 1990, 265, 20, 11734-11739.

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