Difference between revisions of "Part:BBa K1807009"
 
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<partinfo>BBa_K1807009 parameters</partinfo>
 
<partinfo>BBa_K1807009 parameters</partinfo>
 
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===Experimental characterization added by CPU-Nanjing 2022 TEAM===
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<div>
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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.
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[[File:CPU-Nanjing-Parts-PPK-1.png|200px|center]]
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<center>Figure 1. Microscopic observation of polyP granules in E. coli K12/PPK. Intracellular polyP granules appear blue-purple. Scale bar, 2 μm.</center>
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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.
 +
 +
PPK-M is a mutant of E. coli polyphosphate kinase (PPK). Compared to native E. coli PPK that has an alanine and a glutamine residue in position 327 and 328, PPK-M has much more strongly charged glutamate and lysine residues. It has been documented that expression of PPK-M can disrupt intracellular PPK-repressing interactions and thus favor higher levels of polyP accumulation in vivo, as compared to the situation found with expression of PPK. We validated the effectiveness of PPK-M on enhanced intracellular polyP synthesis by comparison with PPK.
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 +
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1. Intracellular polyP content determination
 +
 +
Two engineered E. coli K12 strains that overexpress PPK and PPK-M, respectively, were constructed. Nutrient-poor synthetic municipal wastewater was adopted to evaluate polyP synthesis.
 +
 +
After 12 h of cultivation, intracellular polyP assay showed that the amount of polyP produced by K12/PPK-M was significantly higher than that obtained by K12/PPK (Figure. 1).
 +
[[File:CPU-Nanjing-Parts-PPK-M-21.png|300px|center]]
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<center>Figure 1. Intracellular polyP content of K12/PPK-M compared with K12/PPK.</center>
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2. Microscopic observation
 +
 +
Consistent with quantitative polyP assays, polyP granules observed in K12/PPK-M were significantly larger than those found in K12/PPK (Figure. 2).
 +
[[File:CPU-Nanjing-Parts-PPK-M-311.png|300px|center]]
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<center>Figure 2. Light microscopy images of toluidine blue stained cells. Intracellular polyP granules appear blue-purple. Scale bar, 2 μm.</center>
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==iGEM2021_Nanjing-China Experiment==
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<div>
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<b>Group: Nanjing-China 2021</b>
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<br>
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<b>Author: Hao Yin</b>
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<br><br>
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<p>
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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.
 +
 +
[[File:T--Nanjing-China--ppk1.png|800px|thumb|center|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. 
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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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</p>
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<p>Reference:
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Kyunghye Ahn and Arthur Kornberg. Polyphosphate Kinase from Escherichia coli[J]. The Journal of Biological Chemistry, 1990, 265, 20, 11734-11739.
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</p>
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<!--to here-->
 
<h2>iGEM2019_Nanjing China Experiment</h2>
 
<h2>iGEM2019_Nanjing China Experiment</h2>
 
<p>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.</p>
 
<p>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.</p>
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<p>We test supernatant Pi concentration and optical density of Ecoil and Ecoil+Ecoilppk in Synthetic municipal wastewater(SMW).</p>
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<p>We test supernatant Pi concentration and optical density of Ecoil(DH5a) and DH5a-MDPP in Synthetic municipal wastewater(SMW).</p>
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<p>Ps:
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SMW means Synthetic municipal wastewater</p>
 +
<p>DH5a-MDPP means solo medium-copy DH5a ppk in DH5a</p>
  
−
<p>[[File:T--Nanjing-China--pi.png|800px|thumb|center|Figure 1）supernatant Pi concentration in SMW]] </p>
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<p>[[File:T--Nanjing-China--bronze1.png|800px|thumb|center|Figure 1）supernatant Pi concentration in SMW]] </p>
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<p>[[File:T--Nanjing-China--bronze2.png|800px|thumb|center|Figure 2）optical density of Ecoil in SMW]] </p>

Latest revision as of 12:18, 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.

PPK-M is a mutant of E. coli polyphosphate kinase (PPK). Compared to native E. coli PPK that has an alanine and a glutamine residue in position 327 and 328, PPK-M has much more strongly charged glutamate and lysine residues. It has been documented that expression of PPK-M can disrupt intracellular PPK-repressing interactions and thus favor higher levels of polyP accumulation in vivo, as compared to the situation found with expression of PPK. We validated the effectiveness of PPK-M on enhanced intracellular polyP synthesis by comparison with PPK.


1. Intracellular polyP content determination

Two engineered E. coli K12 strains that overexpress PPK and PPK-M, respectively, were constructed. Nutrient-poor synthetic municipal wastewater was adopted to evaluate polyP synthesis.

After 12 h of cultivation, intracellular polyP assay showed that the amount of polyP produced by K12/PPK-M was significantly higher than that obtained by K12/PPK (Figure. 1).

CPU-Nanjing-Parts-PPK-M-21.png
Figure 1. Intracellular polyP content of K12/PPK-M compared with K12/PPK.


2. Microscopic observation

Consistent with quantitative polyP assays, polyP granules observed in K12/PPK-M were significantly larger than those found in K12/PPK (Figure. 2).

CPU-Nanjing-Parts-PPK-M-311.png
Figure 2. Light microscopy images of toluidine blue stained cells. Intracellular polyP granules appear blue-purple. Scale bar, 2 μm.


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