Difference between revisions of "Part:BBa K5034205"

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===Basic Description===
 
===Basic Description===
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This basic part encodes the PPK2 gene which is initially from Pseudomonas aeruginosa and we performed codon optimization on, is expressed in the PYYDT plasmid. The PPK2 enzyme facilitates the reversible conversion between inorganic polyphosphate (PolyP) and inorganic phosphate (Pi), playing a crucial role in phosphate metabolism. It distinguished from PPK1 by the following: synthesis of poly P from GTP or ATP, a preference for Mn2+ over Mg2+, and a stimulation by Poly P. The forward reaction, a poly P-driven nucleoside diphosphate kinase synthesis of GTP from GDP, is 75-fold greater than the reverse reaction, Poly P synthesis from GTP.
 
  
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In a sentence,It can reversibly convert Poly p and Pi. For the first time, we expressed this element in a strain of Shewanella and conducted codon optimization based on Shewanella.
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This basic part encodes the ''PPK2'' gene which is sourced from ''Pseudomonas aeruginosa'' and we performed codon optimization on it. It is expressed in the pBBR1MCS-terminator plasmid. The PPK2 enzyme facilitates the reversible conversion between inorganic polyphosphate (PolyP) and inorganic phosphate(Pi) with a tendency to produce Pi, playing a crucial role in phosphate metabolism.  
  
 +
It distinguished from PPK1 by the following: (1)It synthesis of poly P from GTP or ATP with a preference for Mn2+ over Mg2+. (2)It is  stimulated by Poly P. (3) The forward reaction, a PolyP-driven nucleoside diphosphate kinase synthesis of GTP from GDP, is 75-fold greater than the reverse reaction, PolyP synthesis from GTP.
  
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In a sentence,It can reversibly convert PolyP and Pi. For the first time, we expressed this element in a strain of ''S. oneidensis'' and conducted codon optimization based on ''S. oneidensis''.
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Figure 1: Basic function of PPK2
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<html>
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<div align="center">
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<img style="width:60%;height:auto;" src="https://static.igem.wiki/teams/5034/engineering/mechanism-of-ppk2.png">
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<p>
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<center>Figure 1: Basic function of PPK2</center>
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</p>
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</div>
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</html>
  
 
===Sequence and Features===
 
===Sequence and Features===
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<partinfo>BBa_K5034205 SequenceAndFeatures</partinfo>
 
  
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===Chassis and Genetic Context===
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In this basic part, only coding sequence is included.But the translational unit is composed of components below.
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Successfully expressed in Escherichia coli DH5α and BL21(DE3) strains.
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Promoter: Constitutive promoter for continuous expression. We use Lac promoter in our experiment.
  
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===Construct features(only coding sequence included in basic part)===
 
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Promoter: Constitutive promoter for continuous expression. We use tac promoter in our experiment.
 
 
RBS: Strong ribosome binding site for efficient translation. We use BBa-B0034 which shows the relatively strongest translation in our experiment.
 
RBS: Strong ribosome binding site for efficient translation. We use BBa-B0034 which shows the relatively strongest translation in our experiment.
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PPK2 Coding Sequence: Encodes the polyphosphate kinase 2 enzyme.
 
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Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use T7Te terminator in our experiment.
 
  
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''PPK2'' Coding Sequence: Encodes the polyphosphate kinase 2 enzyme.
  
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<div class="text-graph">
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Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use a double terminator rrnBT1-T7TE(BBa_B0015) in our experiment.
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https://static.igem.wiki/teams/5034/engineering/fig17.png
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</div>
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<partinfo>BBa_K5034205 SequenceAndFeatures</partinfo>
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<p align="center">
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Figure 2: PCR of target genes before plasmids construction (The extra small fragment in the picture is primer dimer)
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<html>
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<div align="center">
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<img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/results/new/basic-structure-of-ppk2.png">
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<p>
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Figure 2: The translational unit of PPK2
 
</p>
 
</p>
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</div>
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</html>
  
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===Origin (Organism)===
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===Origin===
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Gene Source: Pseudomonas aeruginosa PAO1 strain.
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Gene Source: <i>Pseudomonas aeruginosa</i> PAO1 strain.
  
 
===Experimental Characterization and results===
 
===Experimental Characterization and results===
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In our team’s previous research we found that the behavior of the modified Shewanella did not reach our expectation and the electron microscopic observation also showed an abnormal morphology of the bacterium, we postulated that too much PPK1 may lead to an abnormal charge distribution in the bacterium thus result in a decrease in the bacterium's activity and a reduction in its capacity for electricity production, so we planed to improve the situation by introducing different polyphosphate hydrolases which influence the phosphorus metabolism of Shewanella.
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The results of our amplification of this enzyme are shown in the figure.(Fig.3)
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Electricity production: Using half-cell reaction(electrochemistry) to measure the electricity production ability.
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<html>
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Capacity to polymerize phosphorus: Conducting molybdate assays to determine Pi concentration.
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<div align="center">
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The expression of hydrolase PPK2 showed relatively high phosphorus accumulation and electricity generation ability. Also, the ATP level is considerably enhanced.
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<img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/engineering/fig17.png">
 +
<p>
 +
Figure 3: PCR of target genes before plasmids construction (The extra small fragment in the picture is primer dimer)
 +
</p>
 +
</div>
 +
</html>
 +
When we are conducting the project of the Nanjing China team in 2024,we found that the introduction of PolyP synthase in Shewanella decrease the current significantly. So we have a strategy of introducing PolyP hydrolase, and this element is one of the PolyP hydrolase.  
  
 +
Consistent with our expectations, we found a significant increase in ''S. oneidensis'' current after the introduction of this element (Fig.4).
 +
<html>
 +
<div align="center">
 +
<img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/current.png">
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<p>
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Figure 4: Statistical data on electricity production capacity of <i>S. oneidensis</i> with the introduction of different hydrolases
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</p>
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</div>
 +
</html>
  
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Miraculously, the ability of ''S. oneidensis'' to accumulate phosphorus also increased after the introduction of phosphorus hydrolase (Fig.5).
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<html>
 +
<div align="center">
 +
<img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/engineering/pi-of-ppk2.png">
 +
<p>
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Figure 5: Statistical data on the phosphorus accumulation capacity of <i>S. oneidensis</i> with <i>PPK2</i>
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</p>
 +
</div>
 +
</html>
  
 +
Therefore, we performed ATP and NADH/NAD^+^ measurements (Fig.6). It was found that the introduction of this element could increase the levels of ATP and NADH in the cell, proving that this element could increase the metabolic level of ''S. oneidensis''.
  
 +
<html>
 +
<div align="center">
 +
<img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/results/figure15.png">
 +
<p>
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Figure 6: ATP level and NAD<sup>+</sup>/NADH in <i>S. oneidensis</i> with the introduction of different hydrolases
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</p>
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</div>
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</html>
  
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In a sentence,the expression of hydrolase PPK2 showed relatively high phosphorus accumulation and electricity generation ability because of raising level of metabolism.
  
 +
Experimental manipulation：
  
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<html><a href="https://2024.igem.wiki/nanjing-china/experiments#s16">Electricity production:</a>Using half-cell reaction(electrochemistry) We use half-cell experiment to measure the electricity production ability.</html>
  
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<html><a href="https://2024.igem.wiki/nanjing-china/experiments#s11">Capacity to absorb phosphorus:</a>Conducting molybdate assays to determine Pi concentration.</html>
  
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<html><a href="https://2024.igem.wiki/nanjing-china/experiments#s13">Determination of ATP levels:</a>We use enhanced ATP Assay Kit.</html>
  
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<html><a href="https://2024.igem.wiki/nanjing-china/experiments#s14">Determination of NAD<sup>+</sup>/NADH levels:</a>We use NAD+ /NADH Assay Kit with WST-8.</html>
  
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Figure 3: statistical data on electricity production capacity of Shewanella with the introduction of different hydrolases
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<html><p>More Details of all experiments can be found at the <a href="https://2024.igem.wiki/nanjing-china/experiments">Experiments section on the Wiki.</a></p></html>
  
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===Chassis and Genetic Context===
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Successfully expressed in ''Shewanella onediensis'' MR-1.
  
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Figure 4: statistical data on the phosphorus accumulation capacity of Shewanella with PPK2
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===Potential Applications===
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Managing phosphate levels in contaminated environments;
  
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Enhancing phosphate metabolism in engineered microbial systems;
  
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Optimizing phosphate utilization in industrial microbial processes.
  
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Figure 5: ATP level in Shewanella with the introduction of different hydrolases
 
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===Potential Applications===
 
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Managing phosphate levels in contaminated environments; Enhancing phosphate metabolism in engineered microbial systems; Optimizing phosphate utilization in industrial microbial processes.
 
 
Enhancing the performance of bioelectrochemical systems for electricity generation in providing a renewable and sustainable source of electricity, reducing reliance on fossil fuels and contributing to cleaner energy production.
 
Enhancing the performance of bioelectrochemical systems for electricity generation in providing a renewable and sustainable source of electricity, reducing reliance on fossil fuels and contributing to cleaner energy production.
  
 
===References===
 
===References===
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1.Zhang, H., Ishige, K., & Kornberg, A. (2002). A polyphosphate kinase (PPK2) widely conserved in bacteria. Proceedings of the National Academy of Sciences, 99(26), 16678-16683.  
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<i>1.Zhang, H., Ishige, K., & Kornberg, A. (2002). A polyphosphate kinase (PPK2) widely conserved in bacteria. Proceedings of the National Academy of Sciences, 99(26), 16678-16683. </i>
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2.Neville, N., Roberge, N., & Jia, Z. (2022). Polyphosphate Kinase 2 (PPK2) enzymes: Structure, function, and roles in bacterial physiology and virulence. International Journal of Molecular Sciences, 23(2), 670. 
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3.Itoh, H., & Shiba, T. (2004). Polyphosphate synthetic activity of polyphosphate:AMP phosphotransferase in Acinetobacter johnsonii 210A. Journal of Bacteriology, 186(15), 5178-5181.
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+
  
 +
<i>2.Neville, N., Roberge, N., & Jia, Z. (2022). Polyphosphate Kinase 2 (PPK2) enzymes: Structure, function, and roles in bacterial physiology and virulence. International Journal of Molecular Sciences, 23(2), 670. </i>
  
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<!-- Uncomment this to enable Functional Parameter display
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<i>3.Itoh, H., & Shiba, T. (2004). Polyphosphate synthetic activity of polyphosphate:AMP phosphotransferase in Acinetobacter johnsonii 210A. Journal of Bacteriology, 186(15), 5178-5181.</i>
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===Functional Parameters===
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<partinfo>BBa_K5034205 parameters</partinfo>
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Latest revision as of 09:37, 1 October 2024

PolyP <->Pi

Basic Description

This basic part encodes the PPK2 gene which is sourced from Pseudomonas aeruginosa and we performed codon optimization on it. It is expressed in the pBBR1MCS-terminator plasmid. The PPK2 enzyme facilitates the reversible conversion between inorganic polyphosphate (PolyP) and inorganic phosphate(Pi) with a tendency to produce Pi, playing a crucial role in phosphate metabolism.

It distinguished from PPK1 by the following: (1)It synthesis of poly P from GTP or ATP with a preference for Mn2+ over Mg2+. (2)It is stimulated by Poly P. (3) The forward reaction, a PolyP-driven nucleoside diphosphate kinase synthesis of GTP from GDP, is 75-fold greater than the reverse reaction, PolyP synthesis from GTP.

In a sentence,It can reversibly convert PolyP and Pi. For the first time, we expressed this element in a strain of S. oneidensis and conducted codon optimization based on S. oneidensis.

Figure 1: Basic function of PPK2

Sequence and Features

In this basic part, only coding sequence is included.But the translational unit is composed of components below.

Promoter: Constitutive promoter for continuous expression. We use Lac promoter in our experiment.

RBS: Strong ribosome binding site for efficient translation. We use BBa-B0034 which shows the relatively strongest translation in our experiment.

PPK2 Coding Sequence: Encodes the polyphosphate kinase 2 enzyme.

Terminator: Efficient transcription terminator to ensure proper mRNA processing. We use a double terminator rrnBT1-T7TE(BBa_B0015) in our experiment.


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]

Figure 2: The translational unit of PPK2

Origin

Gene Source: Pseudomonas aeruginosa PAO1 strain.

Experimental Characterization and results

The results of our amplification of this enzyme are shown in the figure.(Fig.3)

Figure 3: PCR of target genes before plasmids construction (The extra small fragment in the picture is primer dimer)

When we are conducting the project of the Nanjing China team in 2024,we found that the introduction of PolyP synthase in Shewanella decrease the current significantly. So we have a strategy of introducing PolyP hydrolase, and this element is one of the PolyP hydrolase.

Consistent with our expectations, we found a significant increase in S. oneidensis current after the introduction of this element (Fig.4).

Figure 4: Statistical data on electricity production capacity of S. oneidensis with the introduction of different hydrolases

Miraculously, the ability of S. oneidensis to accumulate phosphorus also increased after the introduction of phosphorus hydrolase (Fig.5).

Figure 5: Statistical data on the phosphorus accumulation capacity of S. oneidensis with PPK2

Therefore, we performed ATP and NADH/NAD^+^ measurements (Fig.6). It was found that the introduction of this element could increase the levels of ATP and NADH in the cell, proving that this element could increase the metabolic level of S. oneidensis.

Figure 6: ATP level and NAD+/NADH in S. oneidensis with the introduction of different hydrolases

In a sentence,the expression of hydrolase PPK2 showed relatively high phosphorus accumulation and electricity generation ability because of raising level of metabolism.

Experimental manipulation：

Electricity production:Using half-cell reaction(electrochemistry) We use half-cell experiment to measure the electricity production ability.

Capacity to absorb phosphorus:Conducting molybdate assays to determine Pi concentration.

Determination of ATP levels:We use enhanced ATP Assay Kit.

Determination of NAD+/NADH levels:We use NAD+ /NADH Assay Kit with WST-8.

More Details of all experiments can be found at the Experiments section on the Wiki.

Chassis and Genetic Context

Successfully expressed in Shewanella onediensis MR-1.

Potential Applications

Managing phosphate levels in contaminated environments;

Enhancing phosphate metabolism in engineered microbial systems;

Optimizing phosphate utilization in industrial microbial processes.

Enhancing the performance of bioelectrochemical systems for electricity generation in providing a renewable and sustainable source of electricity, reducing reliance on fossil fuels and contributing to cleaner energy production.

References

1.Zhang, H., Ishige, K., & Kornberg, A. (2002). A polyphosphate kinase (PPK2) widely conserved in bacteria. Proceedings of the National Academy of Sciences, 99(26), 16678-16683.

2.Neville, N., Roberge, N., & Jia, Z. (2022). Polyphosphate Kinase 2 (PPK2) enzymes: Structure, function, and roles in bacterial physiology and virulence. International Journal of Molecular Sciences, 23(2), 670. 

3.Itoh, H., & Shiba, T. (2004). Polyphosphate synthetic activity of polyphosphate:AMP phosphotransferase in Acinetobacter johnsonii 210A. Journal of Bacteriology, 186(15), 5178-5181.