Difference between revisions of "Part:BBa K5034205"
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===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 ''S. oneidensis'' 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 ''S. oneidensis''.
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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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Capacity to polymerize phosphorus: Conducting molybdate assays to determine Pi concentration.
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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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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.
  
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<html><p>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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Consistent with our expectations, we found a significant increase in Shewanella current after the introduction of this element (Fig.4).  
 
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Miraculously, the ability of Shewanella to accumulate phosphorus also increased after the introduction of phosphorus hydrolase (Fig.5).
 
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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 Shewanella.
  
 
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<img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/atp.png">
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<img style="width:50%;height:auto;" src="https://static.igem.wiki/teams/5034/results/figure15.png">
 
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Figure 6: ATP level in <i>S. oneidensis</i> with the introduction of different hydrolases
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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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In a sentence,the expression of hydrolase PPK2 showed relatively high phosphorus accumulation and electricity generation ability because of raising level of metabolism.
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Experimental manipulation：
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<html><a href="https://2024.igem.wiki/nanjing-china/experiments#s16">Electricity production:</a></p></html> Using half-cell reaction(electrochemistry) We use half-cell experiment to measure the electricity production ability.
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<html><a href="https://2024.igem.wiki/nanjing-china/experiments#s11">Capacity to absorb phosphorus:</a></p></html> Conducting molybdate assays to determine Pi concentration.
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<html><a href="https://2024.igem.wiki/nanjing-china/experiments#s13">Determination of ATP levels:</a></p></html>We use enhanced ATP Assay Kit.
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<html><a href="https://2024.igem.wiki/nanjing-china/experiments#s14">Determination of NAD<sup>+</sup>/NADH levels:</a></p></html>We use NAD+ /NADH Assay Kit with WST-8.
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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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===Potential Applications===
 
===Potential Applications===

Revision as of 07:32, 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 component 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.

Chassis and Genetic Context

Successfully expressed in Escherichia coli DH5α and BL21(DE3) strains.

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 Shewanella 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 Shewanella 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 Shewanella.

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.


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.