Difference between revisions of "Part:BBa K5034227"

 
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<partinfo>BBa_K5034227 short</partinfo>
 
<partinfo>BBa_K5034227 short</partinfo>
 
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===Basic Description===
 
===Basic Description===
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 reverse reaction, a poly P-driven nucleoside diphosphate kinase synthesis of GTP from GDP, is 75-fold greater than the forward reaction, Poly P synthesis from GTP.
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This plasmid is the expression vector of <i>PPK2</i> gene(BBa_K5034205).
 +
 
 +
This basic part encodes the <i>PPK2</i> gene which is sourced from <i>Pseudomonas aeruginosa</i> and we performed codon optimization on, is expressed in the pBBR1MCS-terminator 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 PolyP-driven nucleoside diphosphate kinase synthesis of GTP from GDP, is 75-fold greater than the reverse reaction, PolyP synthesis from GTP.
  
 
<html>
 
<html>
<img style="width:60%;height:auto;" src="https://static.igem.wiki/teams/5034/engineering/mechanism-of-ppk2.png">
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<div style="text-align: center;">
<p>
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    <img style="width:60%;height:auto;" src="https://static.igem.wiki/teams/5034/engineering/mechanism-of-ppk2.png">  
Figure 1: Basic function of PPK2
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    <p style="text-align: center;">
</p>
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        Figure 1: Basic function of PPK2
 +
    </p>
 +
</div>
 
</html>
 
</html>
  
 
===Sequence and Features===
 
===Sequence and Features===
 
<partinfo>BBa_K5034227 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5034227 SequenceAndFeatures</partinfo>
 
 
===Chassis and Genetic Context===
 
Successfully expressed in Escherichia coli DH5α and BL21(DE3) strains.
 
  
 
===Construct features===
 
===Construct features===
Promoter: Constitutive promoter for continuous expression. We use tac promoter in our experiment.
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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 strongest translation in our experiment.
+
* RBS: Strong ribosome binding site for efficient translation. We use BBa-B0034 which shows the strongest translation in our experiment.
  
PPK2 Coding Sequence: Encodes the polyphosphate kinase 2 enzyme.
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* <i>PPK2</i> 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.
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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.
  
  
 
<html>
 
<html>
<img style="width:60%;height:auto;" src="https://static.igem.wiki/teams/5034/engineering/fig17.png">
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<div style="text-align: center;">
<p>
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    <img style="width:60%;height:auto;" src="https://static.igem.wiki/teams/5034/engineering/fig17.png">  
Figure 2: PCR of target genes before plasmids construction (The extra small fragment in the picture is primer dimer)
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    <p style="text-align: center;">
</p>
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        Figure 2: PCR of target genes before plasmids construction (The extra small fragment in the picture is primer dimer)
 +
    </p>
 +
</div>
 
</html>
 
</html>
  
  
 
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<html>
 
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<div style="text-align: center;">
Figure 3: Basic construction of PPK2 plasmid
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    <img style="width:60%;height:auto;" src="https://static.igem.wiki/teams/5034/results/new/basic-structure-of-ppk2.png">
 
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    <p style="text-align: center;">
 
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        Figure 3: Basic construction of <i>PPK2</i> plasmid
 
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    </p>
 
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</div>
Figure 4: Construction of PPK2 plasmid
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</html>
  
 
<html>
 
<html>
<img style="width:60%;height:auto;" src="https://static.igem.wiki/teams/5034/engineering/colony-pcr.png">
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<div style="text-align: center;">
<p>
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    <img style="width:60%;height:auto;" src="https://static.igem.wiki/teams/5034/engineering/pbbr1mcs-terminator-ppk2.png">  
Figure 5: Bacterial PCR indicating that different plasmids can replicate in Shewanella
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    <p style="text-align: center;">
</p>
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        Figure 4: Construction of <i>PPK2</i> plasmid
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    </p>
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</div>
 
</html>
 
</html>
  
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Plasmid Backbone: pBBR1MCS plasmid, a standard vector used for gene expression in synthetic biology. The plasmid backbone(BBa_K5034201) of this part is a modified version of pBBR1MCS, with a double terminator(BBa_B0015) on it.
 
Plasmid Backbone: pBBR1MCS plasmid, a standard vector used for gene expression in synthetic biology. The plasmid backbone(BBa_K5034201) of this part is a modified version of pBBR1MCS, with a double terminator(BBa_B0015) on it.
 +
  
 
===Experimental Characterization and results===
 
===Experimental Characterization and results===
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.
 
  
Electricity production: Using half-cell reaction(electrochemistry) to measure the electricity production ability.
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We first performed Colony PCR on the target gene in <i>S. oneidensis</i>(fig5), and the length of PPK2 was 1074 base pairs, which was consistent with the results obtained from the experiments.
  
Capacity to polymerize phosphorus: Conducting molybdate assays to determine Pi concentration.
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<html>
 
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<div style="text-align: center;">
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:60%;height:auto;" src="https://static.igem.wiki/teams/5034/engineering/colony-pcr.png">
 +
    <p style="text-align: center;">
 +
        Figure 5: Colony PCR indicating that different plasmids can replicate in <i>S. oneidensis</i>
 +
    </p>
 +
</div>
 +
</html>
  
 +
Then, we determined the electroproduction capacity of S. oneidensis after introduction of the SPPN1 enzyme (e.g. fig6)
  
 
<html>
 
<html>
<img style="width:60%;height:auto;" src="https://static.igem.wiki/teams/5034/current.png">
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<div style="text-align: center;">
<p>
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    <img style="width:60%;height:auto;" src="https://static.igem.wiki/teams/5034/current.png">  
Figure 6: Statistical data on electricity production capacity of Shewanella with the introduction of different hydrolases
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    <p style="text-align: center;">
</p>
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        Figure 6: Statistical data on electricity production capacity of <i>S. oneidensis</i> with the introduction of different hydrolases
 +
    </p>
 +
</div>
 
</html>
 
</html>
 +
 +
We were surprised to find that, similar to after the introduction of NADK, <i>S. oneidensis</i> had a qualitative leap in its ability to produce electricity
 +
 +
Similarly, we determined the ability of <i>S. oneidensis</i> to aggregate phosphorus after its introduction of PPK2(fig7). We also found that it has a superior ability to aggregate phosphorus compared to WT.
  
 
<html>
 
<html>
<img style="width:60%;height:auto;" src="https://static.igem.wiki/teams/5034/engineering/pi-of-ppk2.png">
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<div style="text-align: center;">
<p>
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    <img style="width:60%;height:auto;" src="https://static.igem.wiki/teams/5034/engineering/pi-of-ppk2.png">  
Figure 7: statistical data on the phosphorus accumulation capacity of Shewanella with PPK2  
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    <p style="text-align: center;">
</p>
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        Figure 7: Statistical data on the phosphorus accumulation capacity of <i>S. oneidensis</i> with PPK2  
 +
    </p>
 +
</div>
 
</html>
 
</html>
 +
 +
At the same time, we can also see from the data measuring ATP levels in <i>S. oneidensis</i>(fig8) that PPK2 increased the metabolic strength of <i>S. oneidensis</i>, which is consistent with the results brought about by the introduction of NADK into <i>S. oneidensis</i>.
 +
 
<html>
 
<html>
<img style="width:60%;height:auto;" src="https://static.igem.wiki/teams/5034/engineering/atp-level-with-different-hydrolyases.png">
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<div style="text-align: center;">
<p>
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    <img style="width:60%;height:auto;" src="https://static.igem.wiki/teams/5034/engineering/atp-level-with-different-hydrolyases.png">  
Figure 8: ATP level in Shewanella with the introduction of different hydrolases
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    <p style="text-align: center;">
</p>
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        Figure 8: ATP level in <i>S. oneidensis</i> with the introduction of different hydrolases
 +
    </p>
 +
</div>
 
</html>
 
</html>
 +
 +
All of the above results suggest that PPK2 will be the key enzyme that will be beneficial in enhancing both of these abilities in <i>S. oneidensis</i>, and we have entered it into the next optimisation of the igem cycle.
 +
 +
<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>
 +
 +
 +
===Chassis and Genetic Context===
 +
Chassis:<i>Shewanella oneidensis</i> MR-1.
 +
 +
The gene can be expressed and function properly in <i>S. oneidensis</i>.
 +
  
 
===Potential Applications===
 
===Potential Applications===
 
Managing phosphate levels in contaminated environments;  
 
Managing phosphate levels in contaminated environments;  
  
Enhancing phosphate metabolism in engineered microbial systems;  
+
* Enhancing phosphate metabolism in engineered microbial systems;  
  
Optimizing phosphate utilization in industrial microbial processes.
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* 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===
 
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.  
 
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. 
 
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.
 
3. Itoh, H., & Shiba, T. (2004). Polyphosphate synthetic activity of polyphosphate:AMP phosphotransferase in Acinetobacter johnsonii 210A. Journal of Bacteriology, 186(15), 5178-5181.

Latest revision as of 14:16, 1 October 2024

Pi <-> PolyP

Basic Description

This plasmid is the expression vector of PPK2 gene(BBa_K5034205).

This basic part encodes the PPK2 gene which is sourced from Pseudomonas aeruginosa and we performed codon optimization on, is expressed in the pBBR1MCS-terminator 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 PolyP-driven nucleoside diphosphate kinase synthesis of GTP from GDP, is 75-fold greater than the reverse reaction, PolyP synthesis from GTP.

Figure 1: Basic function of PPK2

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 4981
    Illegal suffix found in sequence at 1
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 4981
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NotI site found at 9
    Illegal NotI site found at 2834
    Illegal NotI site found at 4987
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 4981
    Illegal BglII site found at 3580
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 4981
    Illegal suffix found in sequence at 2
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 4981
    Illegal XbaI site found at 4996
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NgoMIV site found at 562
    Illegal NgoMIV site found at 4244
    Illegal NgoMIV site found at 4527
    Illegal AgeI site found at 402
  • 1000
    COMPATIBLE WITH RFC[1000]

Construct features

  • 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 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.


Figure 2: PCR of target genes before plasmids construction (The extra small fragment in the picture is primer dimer)


Figure 3: Basic construction of PPK2 plasmid

Figure 4: Construction of PPK2 plasmid


Origin (Organism)

PPK2 Gene Source: Pseudomonas aeruginosa PAO1 strain.

Plasmid Backbone: pBBR1MCS plasmid, a standard vector used for gene expression in synthetic biology. The plasmid backbone(BBa_K5034201) of this part is a modified version of pBBR1MCS, with a double terminator(BBa_B0015) on it.


Experimental Characterization and results

We first performed Colony PCR on the target gene in S. oneidensis(fig5), and the length of PPK2 was 1074 base pairs, which was consistent with the results obtained from the experiments.

Figure 5: Colony PCR indicating that different plasmids can replicate in S. oneidensis

Then, we determined the electroproduction capacity of S. oneidensis after introduction of the SPPN1 enzyme (e.g. fig6)

Figure 6: Statistical data on electricity production capacity of S. oneidensis with the introduction of different hydrolases

We were surprised to find that, similar to after the introduction of NADK, S. oneidensis had a qualitative leap in its ability to produce electricity

Similarly, we determined the ability of S. oneidensis to aggregate phosphorus after its introduction of PPK2(fig7). We also found that it has a superior ability to aggregate phosphorus compared to WT.

Figure 7: Statistical data on the phosphorus accumulation capacity of S. oneidensis with PPK2

At the same time, we can also see from the data measuring ATP levels in S. oneidensis(fig8) that PPK2 increased the metabolic strength of S. oneidensis, which is consistent with the results brought about by the introduction of NADK into S. oneidensis.

Figure 8: ATP level in S. oneidensis with the introduction of different hydrolases

All of the above results suggest that PPK2 will be the key enzyme that will be beneficial in enhancing both of these abilities in S. oneidensis, and we have entered it into the next optimisation of the igem cycle.

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


Chassis and Genetic Context

Chassis:Shewanella oneidensis MR-1.

The gene can be expressed and function properly in S. oneidensis.


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.