Difference between revisions of "Part:BBa K3731000"
 
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==iGEM2020_Nanjing-China Experiment==
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==iGEM2021_Nanjing-China Experiment==
 
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<b>Group: Nanjing-China 2020</b>
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<b>Group: Nanjing-China 2021</b>
 
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<b>Author: Guangyu Hu</b>
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<b>Author: Hao Yin</b>
 
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We found that polyP productivity would reach the highest after inoculation for 14 hours and 100ml of synthetic wastewater in a 200ml Erlenmeyer flask has the highest output and the highest conversion efficiency.
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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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PolyP formation is closely associated with ppk1 gene in CPP. This means the cultivation time and volume will affect P consuming a lot. After comparing different cultivation conditions, we found that polyP productivity will reach the highest after inoculation for 14 hours, that is approximately 20mg/L. Besides, 100ml of synthetic wastewater in a 200ml Erlenmeyer flask has the highest output and the highest conversion efficiency. Without bacterial contamination and normal bacterial viability，it can almost convert all inorganic phosphates into polyphosphates.
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[[File:T--Nanjing-China--ppk1.png|800px|thumb|center|Figure1 SDS-PAGE analysis of PPK]]
  
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<h2>iGEM2019_Nanjing China Experiment</h2>
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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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<p>This year our team develops a simple solo medium-copy plasmid-based polyphosphate kinase (PPK1) overexpression strategy for achieving maximum intracellular polyphosphate accumulation.</p>
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<p>We test plasmid-borne ppk1 expression in CPP via qRT-PCR analysis, supernatant Pi concentration and optical density of CPP and CWT in Synthetic municipal wastewater(SMW).</p>
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<p>Ps:
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SMW means Synthetic municipal wastewater</p>
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<p>CPP means solo medium-copy C. f reundii ATCC8090 ppk in C. f reundii ATCC8090</p>
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<p>CWT means wild type C. f reundii ATCC8090</p>
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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>Reference:
 
<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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Latest revision as of 08:55, 14 September 2021


ppk1 in E.Coli BL21

In bacteria, polyP is synthesized by polyphosphate kinase (PPK). PPK consists of PPK1 and PPK2. PPK1 can lengthen the polymer by using the γ-Pi phosphate bond of ATP, and its reversible reaction is to synthesize ATP from ADP and Pi. PPK2 can use polyP to synthesize GTP or ATP reversibly, and mainly synthesize GTP. The ppk1 codes PPK1, which can promote the synthesis(major function) and decomposition(minor function) of polyP with the residue of ATP.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 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


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